Compositions and processes for targeted delivery, expression and modulation of coding ribonucleic acids in tissue

ABSTRACT

Compositions for expressing a polypeptide within a target organ, the composition comprising a delivery particle, and at least a first mRNA sequence complexed with, encapsulated by, or otherwise associated with the delivery particle, wherein the mRNA sequence comprises at least one coding sequence which codes for at least one polypeptide, at least a first untranslated region (UTR) sequence, and at least one micro-RNA (miRNA) binding site sequence, wherein the miRNA binding site sequence is located within, immediately 5′ to, or immediately 3′ to, the first UTR sequence; and wherein the miRNA binding site sequence provides for differential expression of the coding sequence between first and second cell types comprised within the target organ.

RELATED APPLICATIONS

This Application is a continuation of PCT/US18/49772 filed on Sep. 6,2018, which claims the benefit of priority to GB Patent Application Ser.No. 1714430.4, filed Sep. 7, 2017 and U.S. Provisional PatentApplication Ser. No. 62/632,056, filed Feb. 19, 2018, which are allhereby incorporated by reference in their entirety.

FIELD

The present invention relates to messenger ribonucleic acid (mRNA)delivery technologies, typically nanoparticle-based delivery, andmethods of making and using these mRNA delivery technologies in avariety of therapeutic, diagnostic and prophylactic indications. Suchdelivery systems may be used as stand-alone interventions, or incombination with other therapeutic components.

BACKGROUND

Gene therapy is the process of introducing coding polynucleotides intothe cells of a patient in order to treat disease. For example, a mutatedand/or functionless gene can be replaced in target cells by an intactcopy. Gene therapy often relies on viral vectors to introduce codingpolynucleotides into target cells, but other techniques exist to deliverpolynucleotides to cells without the use of viruses. The advantages ofviruses include relatively high possible transfection rates, as well asthe ability to target the virus to particular cell types by control ofthe binding proteins by which viruses enter a target cell. In contrast,non-viral methods of introducing coding polynucleotides into cells canhave problems with low transfection rates, as well as having limitedoptions for targeting expression to particular organs and cell types.However, the nature of viral intervention carries risks of toxicity andinflammation, but also has limited control over the duration and degreeof the expression of the introduced factor.

Tumour therapies based upon biological approaches have advantages overtraditional chemotherapeutics because they can employ numerous diversemechanisms to target and destroy cancers more precisely—e.g. via directcell lysis, cytotoxic immune effector mechanisms and vascular collapseamongst others. As a result, there has been a significant increase inthe number of clinical studies into the potential of such approaches.However due to the diverse range of therapeutic activities, pre-clinicaland clinical study is complex, as multiple parameters may affect theirtherapeutic potential and, hence, defining reasons for treatment failureor methodologies that might enhance the therapeutic activity can bedifficult. Maintaining on-target activities, tumour specificity andreducing side effects is also a major challenge for such experimentaland powerful therapies.

In non-clinical contexts, too, the ability to induce expression of aparticular gene product such as a polypeptide in a particular targettissue or organ is frequently desired. In many situations, a targettissue or organ, will comprise more than one type of cell, and in suchcases it is also frequently desired to express the gene product todifferent degrees in the different cell types—that is, to providedifferential expression in the different cell types. While methods existto introduce polynucleotides in vitro and in vivo, they have the samelimitations as discussed above.

There is therefore a need to further develop methods and compositionsfor delivery of polynucleotide sequences, such as mRNA, to specificorgans and/or tissues, and methods to modulate the expression of thedelivered polynucleotide sequences in specific cells.

SUMMARY

The present invention accordingly provides compositions and methodswhich are capable of delivering expressible messenger RNA (mRNA) tocells in a target organ, and using the cellular system ofmicroRNA-mediated expression modulation to drive differential expressionin different cells, cell types and/or tissues within the target organ.Nanoscale delivery systems comprising the mRNA are used to enabledelivery within the cells of a target organ. By supplying mRNA, theinvention allows controllable and limited exogenous expression ofpolypeptide gene product from the supplied mRNA within different celltypes, for example, cancerous, non-cancerous, diseased or healthy cells.

The invention can be used to enhance or modulate the function of variousadjunct, co-administered or concurrently administered therapies. Forexample, the invention can be used in conjunction with oncolytic viraltherapy, chemotherapy, antibody therapy or radiotherapy. In an example,mRNA coding for factors which increase the efficacy of an oncolyticvirus administered to a patient can be selectively expressed incancerous cells, thus increasing viral lysis of cancer cells whilepreserving non-cancerous and/or healthy cells. This approach can be usedso that attenuated oncolytic viruses are restored to full potency incancerous cells but not in neighbouring non-cancerous or healthy cells.A key advantage of this approach is that it reduces off target effectsand increases the potency of the therapeutic effect, leading toreductions in dosage and associated side effects.

According to a first aspect of the invention, there is provided acomposition for expressing a polypeptide within a target organ, thecomposition comprising a delivery particle, and at least a first mRNAsequence complexed with, encapsulated by, or otherwise associated withthe delivery particle. The mRNA sequence comprises a coding sequencewhich codes for the polypeptide, at least a first untranslated region(UTR) sequence, and at least one micro-RNA (miRNA) binding sitesequence, wherein the miRNA binding site sequence is located within,immediately 5′ to, or immediately 3′ to, the first UTR sequence. ThemiRNA binding site sequence is selected so as to provide fordifferential expression of the coding sequence between first and secondcell types comprised within the target organ.

In an embodiment of the invention, the mRNA sequence may be encapsulatedby the delivery particle. The delivery particles may compriseaminoalcohol lipidoids. In some embodiments the delivery particles aretargeted towards the target organ, and may further comprise one or moretargeting agents selected from: proteins, peptides, carbohydrates,glycoproteins, lipids, small molecules and nucleic acids, where thesetargeting agents associate preferentially with cells in the targetorgan.

In an embodiment of the invention, the miRNA binding site sequencecomprises a plurality of miRNA binding site sequences. The plurality ofmiRNA binding site sequences may comprise greater than two, suitablygreater than three, typically greater than four binding site sequences.The plurality of miRNA binding site sequences may each be substantiallythe same sequence, or may be one or more substantially differentsequences. The plurality of miRNA binding site sequences may bedifferent variants of sequences which are targets for the same miRNAspecies, or for different variants of the same miRNA species. In anembodiment of the invention, the miRNA binding site sequences maycomprise one or more miRNA-122 binding site sequences, includingvariants and homologues thereof.

The different cell types in the target organ may comprise non-neoplasticcells, neoplastic (pre-cancerous or cancerous) cells, and combinationsthereof. In particular, the first and second cell types may be differentselections from the group comprising non-neoplastic cells, a transformedcell phenotype; a pre-cancerous phenotype; and a neoplastic phenotype.The non-neoplastic cells may be considered as healthy cells, oralternatively may include non-healthy (e.g. cirrhotic, inflamed, orinfected) but otherwise non-cancerous cells.

According to an embodiment of the invention, the target organ comprisesat least a first cell phenotype and at least a second cell phenotype;optionally the target organ comprises at least third, fourth, fifth,sixth, seventh and eighth cell phenotypes; suitably the target organcomprises a plurality of cell phenotypes. Where the invention relates toan embodiment comprising a plurality of cell phenotypes, differentialexpression occurs at detectable levels in at least one of the pluralityof cell phenotypes but to a lesser extent or not detectably in the othercell phenotypes.

In embodiments of the invention the target organ comprises first andsecond cell types presenting different miRNA expression patterns. Thetarget organ may be selected from liver, brain, lung, breast orpancreas. The target organ may be liver, in which embodiment both theparticle and the mRNA are adapted to facilitate differential expressionof the coding sequence within cell types or tissues comprised within theliver of a subject patient or animal.

The first UTR sequence may be located 3′ to the coding sequence. Inother embodiments the first UTR sequence may be located 5′ to the codingsequence. In an embodiment of the invention the mRNA sequence furthercomprises a second UTR sequence, which has at least 90% similarity to aUTR sequence found in at least one of the different cell types withinthe target organ. Optionally second UTR sequence, has at least 90%similarity to a UTR sequence in at least one non-diseased cell typewithin the target organ. Optionally second UTR sequence, has at least90% similarity to a UTR sequence in at least one diseased cell typewithin the target organ.

In some embodiments, the polypeptide comprises a therapeutic enhancementfactor. The therapeutic enhancement factor may be selected from: atumour suppressor protein, a programmed cell death protein, an inhibitorof a programmed cell death pathway, a monoclonal antibody or fragment orderivative thereof, a sequence-specific nuclease, an oncolytic viralvirulence factor, a cytokine, a chemokine, a fluorescent marker proteinand combinations thereof. In an embodiment the therapeutic enhancementfactor is an immunomodulatory molecule selected from the groupconsisting of:

-   -   (i) cytokines involved in immune response and inflammation        selected from one or more of: TNF α, TNFβ, IFNα, IFNβ, IFNgamma,        IL1, IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL11, IL12,        CCL2, CCL3, CCL4, CCL5 CXCL 9, and CXCL10;    -   (ii) dendritic cell activators selected from one or more of:        GM-CSF, TLR7 and TLR9;    -   (iii) molecules targeting the following cellular receptors and        their ligands selected from one or more of: CD40, CD40L, CD160,        2B4, Tim-3, GP-2, B7H3 and B7H4;    -   (iv) TGF β inhibitors;    -   (v) T-cell membrane protein 3 inhibitors;    -   (vi) inhibitors of programmed death 1 (PD1), programmed        death-ligand 1 (PDL1), programmed death-ligand 2 (PDL2),        cytotoxic T-lymphocyte antigen 4 (CTLA4), and        lymphocyte-activation gene 3 (LAG3); and    -   (vii) NF-κB inhibitors.

In an embodiment the composition according to the invention may furthercomprise an oncolytic virus. Suitably the virus selected from any one ofthe Groups I-VII of the Baltimore classification of viruses. Optionally,the oncolytic virus is selected from the group comprising one or moreof: Vesicular Stomatitis Virus, Maraba virus, Polio virus, Reovirus,Measles virus, Newcastle disease virus, Coxsackievirus A21, Parvovirus,Herpes Simplex Virus Type 1, and Adenovirus.

In another aspect of the invention, an isolated mRNA sequence forexpressing a polypeptide within a target organ is provided. The sequencecomprises at least one coding sequence which codes for the polypeptide,at least a first untranslated region (UTR) sequence, and at least onemicro-RNA (miRNA) binding site sequence wherein the miRNA binding sitesequence is located within, immediately 5′ to or immediately 3′ to, thefirst UTR sequence. The miRNA binding site sequence allows fordifferential expression of the coding sequence in different cell typeswithin the target organ. Also envisaged in an embodiment of theinvention is a polynucleotide expression construct encoding this mRNAsequence. It is intended that this aspect may further comprise any ofthe features discussed above in relation to the mRNA sequence of otherembodiments of the invention. In another embodiment the polypeptide maycode for a fluorescent marker protein, such as mCherry, as disclosed inSEQ ID NO:3.

In a further aspect of the invention, there is provided a method for thetreatment, prevention, delay of the onset or progression, of cancer oralleviation of a symptom associated with cancer, the method comprisingadministering to a subject in need thereof a composition as discussedaccording to the above aspects and embodiments. The polypeptide, incertain embodiments, may code for a therapeutic enhancement factor, suchas an immunomodulatory molecule or other factors as previouslydescribed.

In a further embodiment of the invention the mRNA comprises a pluralityof coding sequences, which may encode the same or differentpolypeptides.

In an aspect of the invention there is provided a polynucleotideexpression vector construct encoding the mRNA sequence described.Suitably the polynucleotide expression vector comprises a DNA plasmid.

In embodiments of the invention the subject may be human or a non-humananimal. The cancer may be selected from liver, brain, lung, breast orpancreatic cancer. The cancer may be liver cancer, which may suitably behepatocarcinoma, or metastatic liver cancer. The liver cancer may be aprimary cancer, such as hepatocarcinoma or hepatoblastoma, orsecondary/metastatic cancer in the liver. The metastatic cancer may befrom a known or unknown primary solid tumor. The methods may furthercomprise administering a therapy or therapeutic agent to the subjectsuch as chemotherapy, an oncolytic virus, radiotherapy, a biological, anoncolytic virus, a small molecule drug, a adoptive cell therapy (such asCAR-T cell therapy, CAR-NK therapy), and combinations thereof.

In an aspect of the invention, the compositions and compounds asdiscussed according to the above aspects and embodiments are for use inmedicine, suitably for the treatment of cancer. The cancer may be livercancer, which may suitably be a primary cancer, such as ahepatocarcinoma; a hepatoblastoma; a cholangiocarcinoma; a angiosarcoma,or secondary/metastatic cancer in the liver.

In yet a further aspect of the invention, a process is provided formaking a composition comprising a plurality of delivery particles asdescribed herein, the process comprising:

-   -   (i) providing an encapsulating composition;    -   (ii) providing a solution comprising an mRNA sequence, wherein        the mRNA sequence is for expressing a polypeptide within a        target organ, the mRNA sequence comprising: at least one coding        sequence which codes for the polypeptide;    -   at least a first untranslated region (UTR) sequence;    -   at least one micro-RNA (miRNA) binding site sequence;    -   wherein the miRNA binding site sequence is located within,        immediately 5′ to or    -   immediately 3′ to, the first UTR sequence; and wherein the miRNA        binding site sequence allows for differential expression of the        coding sequence in different cell types within the target organ;    -   (iii) combining the encapsulating composition with the solution        comprising the mRNA sequence in order to form a complex between        the encapsulating composition and the mRNA sequence; and    -   (iv) dispersing the complex of (iii) in order to create a        plurality of delivery particles.

Suitably, the encapsulating composition is comprised of an aminoalcohollipidoid, optionally an ethanolic solution comprising C12-200aminoalcohol lipidoids. Typically the plurality of delivery particlescomprises delivery particles having an average diameter of at leastabout 1 nanometres (nm), suitably at least about 30 nm, optionally atleast about 50 nm and at most about 150 nm.

In yet a further aspect of the invention, there is provided a method forthe treatment, prevention, delay of the onset or progression, of canceror alleviation of a symptom associated with cancer, the methodcomprising providing a composition as discussed according to the aboveaspects and embodiments, and administering the composition incombination or concurrently with an oncolytic virus to a subject in needthereof.

In an embodiment, the mRNA sequence codes for a therapeutic agent whichincreases the efficacy of the oncolytic virus. The oncolytic virus mayhave been attenuated by deletion of one or more virulence genes, and themRNA sequence may code for the one or more virulence genes, or anequivalent thereof.

In some embodiments, the oncolytic virus is selected from any one of theGroups I-VII of the Baltimore classification of viruses. The oncolyticvirus may be selected from the group comprising one or more of:Vesicular Stomatitis Virus, Maraba virus, Polio virus, Reovirus, Measlesvirus, Newcastle disease virus, Coxsackievirus A21, Parvovirus, HerpesSimplex Virus Type 1, and Adenovirus. In an embodiment, the oncolyticvirus is a Herpes Simplex Virus, and the mRNA sequence codes for US3,and may comprise SEQ ID NO: 4. In another embodiment, the oncolyticvirus is a Herpes Simplex Virus, and the mRNA sequence codes for ICP6,and may comprise SEQ ID NO: 5.

DRAWINGS

The invention is further illustrated by reference to the accompanyingdrawings in which:

FIG. 1 shows a schematic of a method of administration of a lipidoidencapsulated mRNA composition according to one embodiment of theinvention.

FIG. 2 shows an example of a cloning method to produce DNA synthesisvectors, which vectors were used to produce the mRNA constructsaccording to embodiments of the invention.

FIG. 3 shows three variants of mRNA constructs used in embodiments ofthe invention, and illustrated in FIG. 4, and possible options for theinsertion point of a pair of miRNA binding sequences (here sequencesthat bind to miR-122) within or adjacent to a UTR sequence located 3′ tothe coding sequence.

FIG. 4 shows examples of DNA plasmids, template plasmids, as well assynthesis vectors for producing the mRNA constructs depicted in FIG. 3.

FIGS. 5, 6 and 7 show examples of methods which may be used to produce asynthesis vector for producing the mRNA construct variants as depictedin FIG. 3.

FIG. 8 shows the chemical formulae of examples of constituent compoundsthat can be used in the preparation of delivery particles according toan embodiment of the invention.

FIG. 9A shows a method of preparation of a nanoformulation of deliveryparticles comprising mRNA according to an embodiment of the invention.

FIG. 9B shows the structure of a cross section of a delivery particlecomprising mRNA according to an embodiment of the invention, and furthercomprising the encapsulating constituent compounds depicted in FIG. 8.

FIG. 10A fluorescent microscopy images indicating the results of anexperiment where cells from healthy human hepatocyte culture (HumanPlateable Hepatocytes, HMCPP5), human hepatocarcinoma (Hep3B) and humanhepatoblastoma (HepG2) cells were transfected in vitro with compositionsaccording to embodiments of the invention. Two delivery particles wereadministered: one containing a mRNA encoding the fluorescent proteinmcherry (mRNA-mCh-DMP^(CTx)) and one one containing an mRNA encoding thefluorescent protein mcherry but where differential expression iscontrolled by miRNA-122 content in the target cells(mRNA-mCh-122-DMP^(CTx)).

FIG. 10B shows a quantification of fluorescence intensity after 48 hoursof cells transfected according to the experiment of FIG. 10A. Resultsare shown as means±SD. Statistical significance was determined using thet test. Asterisks indicate statistically significant difference betweenmRNA-mCherry, mRNA-mCherry-122 expression in transfected cells(****p<0.0001, ***p<0.001).

FIG. 11 shows a graph of results from an experiment in which humanhepatocytes (HMCPP5) were transfected either multiple times (MPT) orsingly (ST) with the delivery particles used in FIG. 10 OA. Expressionof mCherry is determined by the level of fluorescence intensity measuredat 24, 28, 72, 96 and 144 hours after transfection. Results are shown asmeans±SD. Statistical significance was determined using the t test.Asterisks indicate statistically significant difference betweenmRNA-mCherry, mRNA-mCherry-122 expression in transfected cells (*p<0.01,**p<0.05).

FIG. 12A shows fluorescent microscopy images indicating the results ofan experiment where healthy mice hepatocytes (AML12 cell line) weretransfected in vitro with the delivery particles used in FIG. 10A withrelative expression levels of mCherry shown at 24 hours posttransfection.

FIG. 12B shows a graph providing quantification of fluorescenceintensity as % pixels counted for the results of FIG. 12A as well as afurther post-transfection time point of 72 hours.

FIG. 13 shows the results of a Western blot in two experiments (denotedRun 1 and Run 2) where human hepatocytes (HMCPP5), human hepatoblastoma(HepG2) and human hepatocarcinoma (Hep3B) cells were transfected with acomposition according to an embodiment of the invention which comprisedan mRNA encoding an exemplary human polypetide of 25 kDa molecular massunder miRNA differential expression control.

FIG. 14 shows the effect of the Herpes Simplex Virus variant R7041 onthe viability of human cells from a model of hepatocarcinoma (Hep3B) andhepatoblastoma (HepG2). The effects of viral application on relativecell viability are shown.

FIG. 15 shows a timetable for an in vitro experiment where human cellsfrom a model of hepatocarcinoma were treated with a composition andmethod according to an embodiment of the invention and then tested viaMTS colorimetric assay.

FIGS. 16A and 16B show the results of in vitro experiments where humancells from a model of hepatoblastoma (FIG. 16 A) and hepatocarcinoma(FIG. 16 B) were treated with virus alone or in combination with acomposition according to an embodiment of the invention following thetimetable of FIG. 15. The composition is a delivery particle comprisingmRNA coding for US3 (US3 mRNA DMP^(CTx)). The effects of the treatmentson cell viability are shown.

FIGS. 17A and 17B show the results of an in vivo experiments using amouse model of human hepatocarcinoma. FIG. 17A shows the tumor growth(Hep3B cells are labelled with luciferase). FIG. 17B shows fluorescentmicroscopy images of the healthy mouse liver using mRNA coding formCherry—no fluorence was detected when using themCherry-DMP^(CTx)-miRNA122 composition.

FIG. 18 shows an immunohistochemistry micrograph result of an in vivoexperiment using the same mouse model as shown in FIG. 17A. A deliveryparticle comprising mRNA coding for US3 (US3 mRNA DMP^(CTx) miRNA-122)is administered via the tail vein and provides differential expressionbetween non-diseased hepatocytes and tumoural liver tissue as evidencedby darker staining for US3 protein in the tumoural tissue. The boundarybetween the tumour tissue and the non-diseased tissue is shown with adashed line.

DETAILED DESCRIPTION

Unless otherwise indicated, the practice of the present inventionemploys conventional techniques of chemistry, molecular biology,microbiology, recombinant DNA technology, and chemical methods, whichare within the capabilities of a person of ordinary skill in the art.Such techniques are also explained in the literature, for example, M. R.Green, J. Sambrook, 2012, Molecular Cloning: A Laboratory Manual, FourthEdition, Books 1-3, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.; Ausubel, F. M. et al. (1995 and periodic supplements;Current Protocols in Molecular Biology, ch. 9, 13, and 16, John Wiley &Sons, New York, N.Y.); B. Roe, J. Crabtree, and A. Kahn, 1996, DNAIsolation and Sequencing: Essential Techniques, John Wiley & Sons; J. M.Polak and James O'D. McGee, 1990, In Situ Hybridisation: Principles andPractice, Oxford University Press; M. J. Gait (Editor), 1984,Oligonucleotide Synthesis: A Practical Approach, IRL Press; and D. M. J.Lilley and J. E. Dahlberg, 1992, Methods of Enzymology: DNA StructurePart A: Synthesis and Physical Analysis of DNA Methods in Enzymology,Academic Press. Each of these general texts is herein incorporated byreference.

Prior to setting forth the invention, a number of definitions areprovided that will assist in the understanding of the invention. Allreferences cited herein are incorporated by reference in their entirety.Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs.

As used herein, the term ‘comprising’ means any of the recited elementsare necessarily included and other elements may optionally be includedas well. ‘Consisting essentially of’ means any recited elements arenecessarily included, elements that would materially affect the basicand novel characteristics of the listed elements are excluded, and otherelements may optionally be included. ‘Consisting of’ means that allelements other than those listed are excluded. Embodiments defined byeach of these terms are within the scope of this invention.

The term ‘isolated’, when applied to a polynucleotide sequence, denotesthat the sequence has been removed from its natural organism of originand is, thus, free of extraneous or unwanted coding or regulatorysequences. The isolated sequence is suitable for use in recombinant DNAprocesses and within genetically engineered protein synthesis systems.Such isolated sequences include cDNAs, mRNAs and genomic clones. Theisolated sequences may be limited to a protein encoding sequence only,or can also include 5′ and 3′ regulatory sequences such as promoters andtranscriptional terminators. Prior to further setting forth theinvention, a number of definitions are provided that will assist in theunderstanding of the invention.

A ‘polynucleotide’ is a single or double stranded covalently-linkedsequence of nucleotides in which the 3′ and 5′ ends on each nucleotideare joined by phosphodiester bonds. The polynucleotide may be made up ofdeoxyribonucleotide bases or ribonucleotide bases. Polynucleotidesinclude DNA and RNA, and may be manufactured synthetically in vitro orisolated from natural sources. Sizes of polynucleotides are typicallyexpressed as the number of base pairs (bp) for double strandedpolynucleotides, or in the case of single stranded polynucleotides asthe number of nucleotides (nt). One thousand bp or nt equal a kilobase(kb). Polynucleotides of less than around 40 nucleotides in length aretypically called ‘oligonucleotides’. The term ‘nucleic acid sequence’ asused herein, is a single or double stranded covalently-linked sequenceof nucleotides in which the 3′ and 5′ ends on each nucleotide are joinedby phosphodiester bonds. The polynucleotide may be made up ofdeoxyribonucleotide bases or ribonucleotide bases. Nucleic acidsequences may include DNA and RNA, and may be manufactured syntheticallyin vitro or isolated from natural sources. Sizes of nucleic acidsequences, also referred to herein as ‘polynucleotides’ are typicallyexpressed as the number of base pairs (bp) for double strandedpolynucleotides, or in the case of single stranded polynucleotides asthe number of nucleotides (nt). One thousand bp or nt equal a kilobase(kb). Polynucleotides of less than around 40 nucleotides in length aretypically called ‘oligonucleotides’ and may comprise primers for use inmanipulation of DNA such as via polymerase chain reaction (PCR).

The term ‘nucleic acid’ as used herein, is a single or double strandedcovalently-linked sequence of nucleotides in which the 3′ and 5′ ends oneach nucleotide are joined by phosphodiester bonds. The polynucleotidemay be made up of deoxyribonucleotide bases or ribonucleotide bases.Nucleic acids may include DNA and RNA, and may be manufacturedsynthetically in vitro or isolated from natural sources. Nucleic acidsmay further include modified DNA or RNA, for example DNA or RNA that hasbeen methylated, or RNA that has been subject to post-translationalmodification, for example 5′-capping with 7-methylguanosine,3′-processing such as cleavage and polyadenylation, and splicing.Nucleic acids may also include synthetic nucleic acids (XNA), such ashexitol nucleic acid (HNA), cyclohexene nucleic acid (CeNA), threosenucleic acid (TNA), glycerol nucleic acid (GNA), locked nucleic acid(LNA) and peptide nucleic acid (PNA). Sizes of nucleic acids, alsoreferred to herein as ‘polynucleotides’ are typically expressed as thenumber of base pairs (bp) for double stranded polynucleotides, or in thecase of single stranded polynucleotides as the number of nucleotides(nt). One thousand bp or nt equal a kilobase (kb). Polynucleotides ofless than around 100 nucleotides in length are typically called‘oligonucleotides’ and may comprise primers for use in manipulation ofDNA such as via polymerase chain reaction (PCR).

In specific embodiments of the present invention the nucleic acidsequence comprises messenger RNA (mRNA).

According to the present invention, homology to the nucleic acidsequences described herein is not limited simply to 100% sequenceidentity. Many nucleic acid sequences can demonstrate biochemicalequivalence to each other despite having apparently low sequenceidentity. In the present invention homologous nucleic acid sequences areconsidered to be those that will hybridise to each other underconditions of low stringency (Sambrook J. et al, supra).

The term ‘operatively linked’, when applied to nucleic acid sequences,for example in an expression construct, indicates that the sequences arearranged so that they function cooperatively in order to achieve theirintended purposes. By way of example, in a DNA vector a promotersequence allows for initiation of transcription that proceeds through alinked coding sequence as far as a termination sequence. In the case ofRNA sequences, one or more untranslated regions (UTRs) may be arrangedin relation to a linked protein coding sequence referred to as an openreading frame (ORF). A given mRNA may comprise more than one ORFs, aso-called polycistronic RNA. A UTR may be located 5′ or 3′ in relationto an operatively linked coding sequence ORF. UTRs may comprisesequences typically found in mRNA sequences found in nature, such asKozak consensus sequences, initiation codons, cis-acting regulatoryelements, poly-A tails, internal ribosome entry sites (IRES), structuresregulating mRNA longevity, sequences directing the localisation of themRNA, and so on. A mRNA may comprise multiple UTRs that are the same ordifferent.

The term ‘expressing a polypeptide’ in the context of the presentinvention refers to production of a polypeptide for which thepolynucleotide sequences described herein code. Typically, this involvestranslation of the supplied mRNA sequence by the ribosomal machinery ofthe cell to which the sequence is delivered.

The term ‘delivery particle’ as used herein refers to particles whichcan comprise therapeutic components by encapsulation, holding within amatrix, the formation of complex or by other means, and deliver atherapeutic component such as a coding nucleic acid sequence into atarget cell. Delivery particles may on the micro-scale, but in specificembodiments may typically be on the nanoscale—i.e. nanoparticles.Nanoparticles are typically sized at least 50 nm (nanometres), suitablyat least approximately 100 nm and typically at most 150 nm, 200 nm,although optionally up to 300 nm in diameter. In one embodiment of theinvention the nanoparticles have a mean diameter of approximately atleast 60 nm. An advantage of these sizes is that this means that theparticles are below the threshold for reticuloendothelial system(mononuclear phagocyte system) clearance, i.e. the particle is smallenough not to be destroyed by phagocytic cells as part of the body'sdefence mechanism. This facilitates the use of intravenous deliveryroutes for the compositions of the invention.

Alternative possibilities for the composition of the nanoparticlesinclude polylactic acid (PLA), poly(lactic-co-glycolic acid) (PLGA), alipid- or phospholipid-based particles such as liposomes; particlesbased on proteins and/or glycoproteins such as collagen, albumin,gelatin, elastin, gliadin, keratin, legumin, zein, soy proteins, milkproteins such as casein, and others (Lohcharoenkal et al. BioMedResearch International; Volume 2014 (2014)); and particles based onmetals or metallic compounds such as gold, silver, aluminium, copperoxides and so on.

In particular, polymers comprising polyethyleneimine (PEI) have beeninvestigated for the delivery of nucleic acids. Nanoparticle vectorscomposed of poly(β-amino esters) (PBAEs) have also been shown to besuitable for nucleic acid delivery, especially in coformulation withpolyethylene glycol (PEG) (Kaczmarek J C et al Angew Chem Int Ed Engl.2016; 55(44): 13808-13812). Particles of such coformulations have beenused to deliver mRNA to the lung.

Also considered are particles based on polysaccharides and theirderivatives, such as cellulose, chitin, and chitosan. Chitosan is acationic linear polysaccharide obtained by partial deacetylation ofchitin, with nanoparticles comprising this substance possessingpromising properties for drug delivery such as biocompatibility, lowtoxicity and small size (Felt et al., Drug Development and IndustrialPharmacy, Volume 24, 1998-Issue 11). It is envisioned that combinationsbetween the above constituents may be used.

US2010/0331234, US2011/0293703 and US2015/0203439—which are incorporatedherein by reference—describe the production of aminoalcohol lipidoids byreacting an amine with an epoxide-terminated compound. Complexes,micelles, liposomes and particles, including nanoparticles, may beprepared with these lipidoids and their chemical structure makes themparticularly suited to the delivery of a ‘cargo’—e.g. nucleic acids suchas coding mRNAs—to target cell types within the body of a human oranimal subject. Delivery platforms comprising aminoalcohol lipidoidcompounds are particularly suitable for use in the delivery of netnegatively charged cargo molecules given the tertiary amines availablefor protonation thus forming a cationic moiety. For example,aminoalcohol lipidoid compounds may be used in the preparation ofparticulate compositions to deliver DNA, RNA, or other polynucleotidecargoes to a subject or to a target cell or tissue. Suitable particlesmay be in the form of microparticles, nanoparticles, liposomes, ormicelles.

The aminoalcohol lipidoid based delivery particles possess tertiaryamines that are available to interact with a polynucleotide cargo, suchas a coding mRNA. Polynucleotides, or derivatives thereof, are contactedwith the aminoalcohol lipidoid compounds under conditions suitable toform polynucleotide/lipidoid complexes. The lipidoid is preferably atleast partially protonated so as to form a complex with the negativelycharged polynucleotide. In this way, the polynucleotide/lipidoidcomplexes form particles that are useful in the delivery of cargopolynucleotides to cells and tissues. In certain embodiments, multipleaminoalcohol lipidoid molecules may be associated with a polynucleotidemolecule. The complex may include at least 1, at least 5, at least 10,at least 20, at least 50, or suitably at least 100 aminoalcohol lipidoidmolecules. The complex may include at most 10,000, at most 5000, at most2000, at most 1000, at most 500, or typically at most 100 aminoalcohollipidoid molecules.

Those of ordinary skill in the art will appreciate that a population ofparticles follow principles of particle size distribution. Widely used,art-recognized methods of describing particle size distributionsinclude, for example, average diameters and D values, such as the D50value, which is commonly used to represent the mean diameter of therange of the particle sizes of a given sample. In certain embodiments,the diameter of the nanoparticles particles ranges from 10-500 nm, moresuitably the diameter of the particles ranges from 10-1200 nm, andparticularly from 50-150 nm. In some embodiments, the nanoparticles haveaverage diameters of at least about 10 nm, suitably at least about 30nm. In some embodiments, nanoparticles have average diameters of lessthan about 150 nm in average diameter and greater than 50 nm in average.

The particles may be further associated with a targeting agent atfacilitates binding of the delivery particle to a target cell type. Theterm ‘targeted’ as used herein in relation to refers to an object, orcomposition such as comprising a delivery particle, which is intended toassociate with and facilitate transfection of cells within a particularorgan, tissue or cell type within the body. In a particular embodiment,a delivery particle—such as a delivry nanoparticle—may be targeted todeliver its cargo only to a certain organ, tissue or cell type.Targeting may be geographical, for example by the delivery of thetargeted object directly to a particular tissue, or may be mediatedchemically, through targeting agents or binding moieties whichpreferentially associate with target cells or tissues.

A variety of targeting agents that direct pharmaceutical compositions toparticular cells are known in the art (see, for example, Cotten et al.Methods Enzym. 217:618, 1993; Wagner et al. Advanced Drug DeliveryReviews, Volume 14, Issue 1, April-May 1994, 113-135; Fiume et al.Advanced Drug Delivery Reviews, Volume 14, Issue 1, April-May 1994,51-65). The targeting agents may be included throughout the particle ormay be localised only on the surface. The targeting agent may be aprotein, peptide, carbohydrate, glycoprotein, lipid, small molecule,nucleic acids, etc. The targeting agent may be used to target specificcells or tissues or may be used to promote endocytosis or phagocytosisof the particle. Examples of targeting agents include, but are notlimited to, antibodies, fragments of antibodies, low-densitylipoproteins (LDLs), transferrin, asialoglycoproteins, gp120 envelopeprotein of the human immunodeficiency virus (HIV), carbohydrates,receptor ligands, sialic acid, aptamers etc. If the targeting agent isdistributed throughout the particle, the targeting agent may be includedin the mixture or composite that is used to form the particles. If thetargeting agent is only located on the surface, the targeting agent maybe associated with the formed particles using standard chemicaltechniques e.g. by covalent binding, hydrophobic, hydrogen bonding, vander Waals, biotin-avidin linkage, or other interactions.

The particulate compositions of certain embodiments of the invention maysuitably deliver the encapsulated mRNA cargo over a period of time thatmay be controlled by the particular choice or formulation of theencapsulating biodegradable non-toxic polymer or biocompatible material.For example, the particulate compositions may release the encapsulatedmRNA cargo over at least 30 minutes, at least 1 hour, at least 2 hours,at least 6 hours, at least 12 hours, or at least 1 day. The particulatecompositions may release the encapsulated mRNA cargo over at most 2days, at most 3 days, or at most 7 days.

The term ‘diseased’ as used herein, as in ‘diseased cells’ and/or‘diseased tissue’ indicates tissues and organs (or parts thereof) andcells which exhibit an aberrant, non-healthy or disease pathology. Forinstance, diseased cells may be infected with a virus, bacterium, prionor eukaryotic parasite; may comprise deleterious mutations; and/or maybe cancerous, precancerous, tumoural or neoplastic. Diseased cells maycomprise an altered intra-cellular miRNA environment when compared tootherwise normal or so-called healthy cells. In certain instancesdisease cells may be pathologically normal but comprise an alteredintra-cellular miRNA environment that represents a precursor state todisease. Diseased tissues may comprise healthy tissues that have beeninfiltrated by diseased cells from another organ or organ system. By wayof example, many inflammatory diseases comprise pathologies whereotherwise healthy organs are subjected to infiltration with immune cellssuch as T cells and neutrophils. By way of a further example, organs andtissues subjected to stenotic or cirrhotic lesions may comprise bothhealthy and diseased cells in close proximity.

The term ‘cancer’ as used herein refers to neoplasms in tissue,including malignant tumours which may be primary cancer starting in aparticular tissue, or secondary cancer having spread by metastasis fromelsewhere. The terms cancer, neoplasm and malignant tumours are usedinterchangeably herein. Cancer may denote a tissue or a cell locatedwithin a neoplasm or with properties associated with a neoplasm.Neoplasms typically possess characteristics that differentiate them fromnormal tissue and normal cells. Among such characteristics are included,but not limited to: a degree of anaplasia, changes in morphology,irregularity of shape, reduced cell adhesiveness, the ability tometastasize, and increased cell proliferation. Terms pertaining to andoften synonymous with ‘cancer’ include sarcoma, carcinoma, malignanttumour, epithelioma, leukaemia, lymphoma, transformation, neoplasm andthe like. As used herein, the term ‘cancer’ includes premalignant,and/or precancerous tumours, as well as malignant cancers.

The term ‘healthy’ as used herein, as in ‘healthy cells’ and/or ‘healthytissue’ indicates tissues and organs (or parts thereof) and cells whichare not themselves diseased and approximate to a typically normalfunctioning phenotype. It can be appreciated that in the context of theinvention the term ‘healthy’ is relative, as, for example,non-neoplastic cells in a tissue affected by tumours may well not beentirely healthy in an absolute sense. Therefore ‘non-healthy cells’ isused mean cells which are not themselves neoplastic, cancerous orpre-cancerous but which may be cirrhotic, inflamed, or infected, orotherwise diseased for example. Similarly, ‘healthy or non-healthytissue’ is used to mean tissue, or parts thereof, without tumours,neoplastic, cancerous or pre-cancerous cells; or other diseases asmentioned above; regardless of overall health. For instance, in thecontext of an organ comprising cancerous and fibrotic tissue, cellscomprised within the fibrotic tissue may be thought of as relatively‘healthy’ compared to the cancerous tissue.

In an alternative embodiment, the health status of a cell, cell type,tissue and/or organ is determined by the quantification of miRNAexpression. In certain disease types, such as cancer, the expression ofparticular miRNA species is affected, and can be up- or down-regulatedcompared to unaffected cells. This difference in the miRNA transcriptomecan be used to identify relative states of health, and/or to track theprogression of healthy cells, cell types, tissues and/or organs towardsa disease state. The disease state may include the various stages oftransformation into a neoplastic cell. In embodiments of the presentinvention the differential variations in the miRNA transcriptome of celltypes comprised within a given organ or organ system is leveraged inorder to control protein expression in the different cell types.

As used herein, the term ‘organ’ is synonymous with an ‘organ system’and refers to a combination of tissues and/or cell types that may becompartmentalised within the body of a subject to provide a biologicalfunction, such as a physiological, anatomical, homeostatic or endocrinefunction. Suitably, organs or organ systems may mean a vascularizedinternal organ, such as a liver or pancreas. Typically organs compriseat least two tissue types, and/or a plurality of cell types that exhibita phenotype characteristic of the organ.

The term ‘therapeutic virus’ as used herein refers to a virus which iscapable of infecting and killing cancer cells, sometimes by direct virallysis (oncolysis), but also including indirect killing by thestimulation of host anti-tumoural responses. Oncolytic viruses arefrequently characterised by having increased activity in diseased cells,including cancer cells, compared with healthy cells.

Examples of oncolytic viruses include those provided in Table 1, andsubtypes thereof.

TABLE 1 Oncolytic virus Type Vesicular Somatitis Virus Enveloped RNAMaraba virus Enveloped rhabdovirus Polio virus Non enveloped RNAReovirus Non enveloped RNA Measles virus Enveloped RNA Newcastle diseasevirus Enveloped RNA Coxsackievirus A21 Non enveloped RNA Parvovirus Nonenveloped DNA Herpes Simplex Virus Type 1 Enveloped DNA Adenovirus Nonenveloped DNA

In embodiments of the invention viruses may be selected from any one ofthe Groups I-VII of the Baltimore classification of viruses (Baltimore D(1971). “Expression of animal virus genomes”. Bacteriol Rev. 35 (3):235-41). In specific embodiments of the invention suitable viruses maybe selected from Baltimore Group I, which are characterised as havingdouble stranded DNA viral genomes; Group IV, which have single strandedpositive RNA genomes; and Group V, which have single stranded negativeRNA genomes.

The term ‘virulence gene’ or ‘virulence factor’ as used herein refers toa gene or gene product which aids in the replication of a therapeuticvirus such as an oncolytic virus within or lysis of the cells which itinfects. The term ‘replication factor’ is used as a synonymous termherein. Virulence factors may typically be viral genes encoded by theviral genome. Virulence factors may be involved in functions such asintracellular immune system suppression and evasion, viral genomereplication, the spread or transmission of virions, the production orassembly of structural coat proteins, the activation of viruses in alatent state, the prevention of viral latency, and the takeover of hostcell processes. Several virulence factors have cellular or otherequivalents which can compensate for the function of these genes iflacking in the virus genome. Some viruses can be modified with exogenousvirulence genes which increase their ability to replicate, lyse cells,and spread.

In specific embodiments of the present invention the compositionsenhance or sustain the oncolytic potency of a virus in a tumor locatedwithin an organ through differential expression of protein orpolypeptide that enhances virion replication preferentially in thetumor. In further embodiments of the invention the compositions mayencode a gene product that controls the interaction between host immunecells and oncolytic virus within a tumour. In yet a further embodiment,the compositions of the invention can be used to produce gene productsthat modulate differential patterns of oncolytic virus activity as wellas expression of immune co-stimulatory molecules that are administeredvia the virion, exogenously or via a delivery particle of the invention.

The term ‘polypeptide’ as used herein is a polymer of amino acidresidues joined by peptide bonds, whether produced naturally or in vitroby synthetic means. Polypeptides of less than around 12 amino acidresidues in length are typically referred to as “peptides” and thosebetween about 12 and about 30 amino acid residues in length may bereferred to as “oligopeptides”. The term “polypeptide” as used hereindenotes the product of a naturally occurring polypeptide, precursor formor proprotein. Polypeptides can also undergo maturation orpost-translational modification processes that may include, but are notlimited to: glycosylation, proteolytic cleavage, lipidization, signalpeptide cleavage, propeptide cleavage, phosphorylation, and such like.The term “protein” is used herein to refer to a macromolecule comprisingone or more polypeptide chains.

The term ‘gene product’ as used herein refers to the product of thecoding sequence or ORF comprised within an mRNA construct as describedherein. The gene product may comprise a polypeptide or a protein. Apolycistronic mRNA construct may result in the production of multiplegene products.

Delivery of mRNA directly to cells allows direct and controllabletranslation of the desired gene products such as polypeptides and/orproteins in the cells. Provision of mRNA specifically allows not onlyfor the use of cell expression modulation mechanisms such as miRNAmediated control (as detailed in specific embodiments below), but alsorepresents a finite and exhaustible supply of the product, rather thanthe potentially permanent change to the transcriptome of a target cellwhich an episomal or genomically inserted DNA vector might provide.

In embodiments of the present invention an mRNA sequence is providedthat comprises a sequence that codes for at least one polypeptide inoperative combination with one or more untranslated regions (UTRs) thatmay confer tissue specificity, and stability to the nucleic acidsequence as a whole. By ‘tissue specificity’ it is meant thattranslation of the protein product encoded by the mRNA is modulatedaccording to the presence of the UTR. Modulation may include permitting,reducing or even blocking detectable translation of the mRNA into aprotein product. The UTRs may be linked directly to the mRNA in cis—i.e.on the same polynucleotide strand. In an alternative embodiment, a firstsequence that codes for a gene product is provided and a further secondsequence, that hybridises to a portion of the first sequence, isprovided that comprises one or more UTRs that confer tissue specificityto the nucleic acid sequence as a whole. In this latter embodiment theUTR is operatively linked to the sequence that encodes the gene productin trans.

According to specific embodiments of the invention, an mRNA is providedthat comprises such associated nucleic acid sequences operatively linkedthereto as are necessary to prevent or reduce expression of a geneproduct in non-diseased liver tissue, e.g. in healthy hepatocytes. Assuch, an mRNA construct, or transcript, is provided that comprises a 5′cap and UTRs necessary for ribosomal recruitment and tissue specificexpression (typically, but not exclusively positioned 3′ to the ORF), aswell as start and stop codons that respectively define the ORF. When theconstruct is introduced into a non-diseased liver, expression of thegene product is prevented or reduced. In contrast, neoplastic cellscomprised within the liver typically do not conform to normalnon-diseased liver cell expression patterns, posessing a quite differentmiRNA transcriptome. The gene product is translated specifically inthese cancer cells but not in neighboring healthy hepatocytes. Deliveryof the mRNA construct to the liver tissue may be achieved via aparticulate delivery platform as described herein. Cell type specificexpression can be mediated via microRNA modulation mechanisms such asthose described in more detail below.

A ‘therapeutic component’ or ‘therapeutic agent’ as defined hereinrefers to a molecule, substance, cell or organism that when administeredto an individual human or other animal as part of a therapeuticintervention, contributes towards a therapeutic effect upon thatindividual human or other animal. The therapeutic effect may be causedby the therapeutic component itself, or by another component of thetherapeutic intervention. The therapeutic component may be a codingnucleic acid component, in particular an mRNA. The coding nucleic acidcomponent may code for a therapeutic enhancement factor, as definedbelow. A therapeutic component may also comprise a drug, optionally achemotherapeutic drug such as a small molecule or monoclonal antibody(or fragment thereof). In some embodiments, a therapeutic component maycomprise a cell, such as a recombinantly modified immune effectorcell—e.g. a CAR-T cell. In other embodiments of the invention, thetherapeutic agent comprises a therapeutic virus, such as an oncolyticvirus or a viral vector.

The term ‘therapeutic effect’ refers to a local or systemic effect in ananimal subject, typically a human, caused by a pharmacologically ortherapeutically active agent that comprises a substance, molecule,composition, cell or organism that has been administered to the subject,and the term ‘therapeutic intervention’ refers to the administration ofsuch a substance, molecule, composition, cell or organism. The term thusmeans any agent intended for use in the diagnosis, cure, mitigation,treatment or prevention of disease or in the enhancement of desirablephysical or mental development and conditions in an animal or humansubject. The phrase ‘therapeutically-effective amount’ means that amountof such an agent that produces a desired local or systemic effect at areasonable benefit/risk ratio applicable to any treatment. In certainembodiments, a therapeutically effective amount of an agent will dependon its therapeutic index, solubility, and the like. For example, certaintherapeutic agents of the present invention may be administered in asufficient amount to produce a reasonable benefit/risk ratio applicableto such treatment. In the specific context of treatment of cancer, a‘therapeutic effect’ can be manifested by various means, including butnot limited to, a decrease in solid tumour volume, a decrease in thenumber of cancer cells, a decrease in the number of metastases observed,an increase in life expectancy, decrease in cancer cell proliferation,decrease in cancer cell survival, a decrease in the expression of tumourcell markers, and/or amelioration of various physiological symptomsassociated with the cancerous condition.

In one embodiment, the subject to whom therapy is administered is amammal (e.g., mouse, rat, primate, non-human mammal, domestic animal orlivestock, such as a dog, cat, cow, horse, sheep, goat and the like),and is suitably a human. In a further embodiment, the subject is ananimal model of cancer. For example, the animal model can be anorthotopic xenograft animal model of a human-derived cancer, suitablyliver cancer.

In a specific embodiment of the methods of the present invention, thesubject has not yet undergone a therapeutic treatment, such astherapeutic viral therapy, chemotherapy, radiation therapy, targetedtherapy, and/or anti-immune checkpoint therapy. In still anotherembodiment, the subject has undergone a therapeutic treatment, such asthe aforementioned therapies.

In further embodiments, the subject has had surgery to remove cancerousor precancerous tissue. In other embodiments, the cancerous tissue hasnot been removed, for example, the cancerous tissue may be located in aninoperable region of the body, such as in a tissue that if subjected tosurgical intervention may compromise the life of the subject, or in aregion where a surgical procedure would cause considerable risk ofpermanent harm.

In some embodiments, the provided mRNA may code for a ‘therapeuticenhancement factor’. According to the present invention therapeuticenhancement factors are gene products or polypeptides that may enhanceor facilitate the ability of another, co-administered therapeutic agent,to exert a therapeutic effect upon a given cell, suitably the targetcell. When introduced into or in the vicinity of the target cell,expression of the therapeutic enhancement factor may cooperate with aco-administered therapeutic agent thereby enabling or enhancing thetherapeutic activity of the agent. In some embodiments, the therapeuticenhancement factor may enhance the ability of a co-administeredoncolytic virus to lyse cancer cells. In other embodiments of theinvention, the therapeutic enhancement factor may effect an alterationof a tumour microenvironment so as to assist or recruit the subject'sown immune response. In this latter embodiment, the alteration of thetumour microenvironment may assist co-administration of an oncolyticvirus or a CAR-T or other adoptive cell based therapy. In someembodiments, the therapeutic enhancement factor may enable theconversion of a prodrug into an active form.

Multiple therapeutic enhancement factors may be combined in compositionsaccording to specific embodiments of the present invention. In suchembodiments, the coding sequences for each therapeutic enhancementfactor may be present in separate mRNA molecules. In some embodiments,sequences for more than one therapeutic enhancement factor may bepresent on the same mRNA molecule. In such cases the polycistronic mRNAmolecule further comprises sequences as necessary for the expression ofall coded sequences, such as internal ribosome entry sites (IRES).

In embodiments where multiple different mRNA molecules are comprised inone or more delivery particles, it is contemplated that each deliveryparticle may comprise one or more than one type of mRNA molecule; thatis, not every delivery particle in a particular embodiment willnecessarily comprise all of the mRNA molecules provided in saidembodiment.

The mRNA constructs of certain embodiments of the invention may besynthesised from a polynucleotide expression construct, which may be forexample a DNA plasmid. This expression construct may comprise anypromoter sequence necessary for the initiation of transcription and acorresponding termination sequence, such that transcription of the mRNAconstruct can occur. Such polynucleotide expression constructs arecontemplated to comprise embodiments of the invention in their ownright.

The gene product encoded by the mRNA is typically a peptide, polypeptideor protein. Where a particular protein consists of more than onesubunit, the mRNA may code for one or more than one subunit.

The gene product encoded by the mRNA may be of any type suitable forproducing a therapeutic effect. In the context of treating cancer, thegene product encoded by the mRNA may suitably include genes which whenexpressed by a cancer cell cause or aid in the destruction of the cancercell.

Tumour suppressor genes such as p53 may be provided by the constructs ofthe invention. p53 plays a role in cell processes including apoptosisand genomic stability. It is involved in the activation of the DNArepair process in response to genomic damage, and can arrest cell growthand reproduction.

Genes which promote cell death by apoptosis—so-called suicidegenes—which when expressed cause the cell to activate the process ofapoptosis, may also be provided by the compositions and constructs ofthe invention. Cancer cells often possess mutated and/or functionlessversions of these apoptosis-related genes, and so cannot undergoapoptosis in response to external signals. Suicide gene therapy may alsorefer to the introduction of genes which allow the conversion of anon-toxic compound or prodrug into a lethal drug (Duarte et al. CancerLetters, 2012). According to embodiments of the invention, such geneproducts can be introduced selectively into diseased cells, such asneoplastic cells, marking them for destruction by induced apoptosis ordelivery of an otherwise non-toxic compound or prodrug.

In specific embodiments of the invention, the mRNA may encode inhibitorsof the programmed cell death pathway, such as inhibitors of PD-1receptor (CD279) or its ligands PD-L1 (B7-H1; CD274) and PD-L2 (B7-DC;CD273). Hence, the mRNA may encode a protein or polypeptide that bindsto or otherwise interferes with the function of the PD-1/PDL-1 orPD-1/PDL-2 axis within diseased or neoplastic cells within a targetorgan. Suitable proteins or polypeptides may include antibodies, whichmay be monoclonal or polyclonal, or antigen binding fragments thereof,or other antigen binding microproteins, that bind to PD-1 receptor,PDL-1, PDL-2, or complexes of ligand and receptor. This effect may alsobe observed by use of protein or polypeptide inhibitors of the cytotoxicT lymphocyte antigen 4 (CTLA4) pathway, another so-called immunecheckpoint. Inhibition of either or both pathways is known to result ina change in the immune response within the tumour microenvironment thatmay positively benefit the health of the patient. In addition, bymodulating the immune response in a subject the compositions of thepresent invention may show particular utility in combinatorial therapieswith other anti-cancer therapeutic approaches, such as radiotherapy orchemotherapy. FDA approved anti-PD1 pathway inhibitors includepembrolizumab and nivolumab. Known anti-PDL-1 inhibitors includeMPDL-3280A, BMS-936559 and atezolizumab. Anti-CTLA4 therapeuticinhibitors include ipilimumab and tremelimumab. The compositions of theinvention may be used to deliver such inhibitors of the programmed celldeath pathway selectively to diseased cells within a target organ in asubject by leveraging the differential miRNA environment in those cells

Chimeric antigen receptor T-cells (CAR-T cells) are immune cells,typically T-lymphocytes, which have been modified to express receptorswhich target cancer cells.

Adoptive immunotherapy, which involves the transfer of autologousantigen-specific T cells generated ex vivo, is a promising strategy totreat viral infections and cancer. The T cells used for adoptiveimmunotherapy can be generated either by expansion of antigen-specific Tcells or redirection of T cells through genetic engineering (see e.g.,Park, T. S., S. A. Rosenberg, et al. (2011). “Treating cancer withgenetically engineered T cells.” Trends Biotechnol 29(11): 550-7).

Novel specificities in T cells, also known as immune effector cells,have been successfully generated through the genetic transfer oftransgenic T cell receptors or chimeric antigen receptors (CARs) (seee.g., Jena, B., G. Dotti, et al. (2010). “Redirecting T-cell specificityby introducing a tumor-specific chimeric antigen receptor.” Blood116(7): 1035-44). CARs are synthetic receptors consisting of at leastthree parts: an extracellular antigen recognition domain (also known asthe ectodomain), a transmembrane domain, and an intracellular T-cellactivation domain (also known as the endodomain). In some embodiments,the engineered T cells comprise a specific class of T cells, such as,for example, gamma delta T cells, a subtype of T cells that selectivelytarget tumoral cells without affecting healthy ones. CARs havesuccessfully allowed T cells to be redirected against antigens expressedat the surface of tumor cells from various malignancies includinglymphomas and solid tumors (Jena, Dotti et al. supra). In someembodiments, the engineered T cells comprise at least a population ofautologous T cells in which the CAR-T cells are engineered to eliminateexpression of the endogenous αβ T-cell receptor (TCR) to prevent agraft-versus-host response without compromising CAR-dependent effectorfunctions. In some embodiments, the engineered T cells comprise at leasta population of allogeneic T cells. In some embodiments, the engineeredT cells comprise at least a population of autologous T cells and apopulation of allogeneic T cells.

Generally, the extracellular antigen recognition domain is a targetingmoiety that is associated with one or more signaling domains in a singlefusion molecule from an antibody, receptor, or ligand domain that bindsa specific target, typically a tumor-associated target. In someembodiments, the extracellular antigen recognition domain is or isderived from a single-chain Fragment variant (scFv) of anantigen-binding domain of a single-chain antibody (scFv), comprising thelight and heavy variable fragments of a monoclonal antibody joined by aflexible linker. In some embodiments, In some embodiments, extracellularantigen recognition domain is linked to the transmembrane domain by alinker, such as, for example, a flexible linker such as the IgG1 hingelinker. In some embodiments, the transmembrane is or is derived from aCD28 transmembrane domain. In some embodiments, the endodomain includesa co-stimulatory domain designed to enhance the immune response, forexample, by enhancing survival and increasing proliferation of CARmodified T cells, and an internal T-cell activation domain designed toactivate the T cell when it binds to the desired target. In someembodiments, the costimulatory domain is or is derived from a CD28costimulatory domain, an OX-40 (CD134) costimulatory domain, an ICOScostimulatory domain, a 4-1BB (CD137) costimulatory domain, or anycombination thereof. In some embodiments, the intracellular T-cellactivation domain comprises the CD3 zeta (CD3ζ) domain or a biologicallyactive portion thereof. In some embodiments, T cell activation resultsin immune cell activation in which inflammatory cytokines are releasedby the T cells to promote an inflammation and/or immune response. Insome embodiments, T cell activation results in cytotoxic activity inwhich cytotoxins are released by the T cells to promote cancer cellapoptosis. In some embodiments, T cell activation results inproliferation in which interleukins are released by the T cells topromote cell development and division. In some embodiments, T cellactivation results in a combination of at least two of immune cellactivation, cytotoxic activity, and/or proliferation.

In some embodiments, the extracellular antigen recognition domainspecifically binds to CD19. CD19 is an attractive target forimmunotherapy because the vast majority of B-acute lymphoblasticleukemia (B-ALL) uniformly express CD19, whereas expression is absent onnon-hematopoietic cells, as well as myeloid, erythroid, and T cells, andbone marrow stem cells. Clinical trials targeting CD19 on B-cellmalignancies are underway with encouraging anti-tumor responses. Many ofthe current CAR-T therapies being evaluated in clinical trials use Tcells genetically modified to express a chimeric antigen receptor (CAR)with specificity derived from the scFv region of a CD19-specific mousemonocional antibody FMC63 (see e.g., Nicholson, Lenton et al. (1997).“Construction and characterisation of a functional CD19 specific singlechain Fv fragment for immunotherapy of B lineage leukaemia andlymphoma.” Mol Immunol. 1997 November-December; 34(16-17):1157-65;Cooper, Topp et al. (2003). “T-cell clones can be rendered specific forCD19: toward the selective augmentation of the graft-versus-B-lineageleukemia effect.” Blood. 2003 Feb. 15; 101(4):1637-44; Cooper, Jena etal. (2012) (International application: WO2013/126712).

In some embodiments, extracellular antigen recognition domainspecifically binds to CD22. CD22 is a transmembrane phosphoglycoproteinthat belongs to the Siglec family of lectins and specifically bindssialic acid with its N-terminus seven extracellular immunoglobulindomains. It mainly acts as an inhibitory receptor for B cell activationand signaling and regulates the interaction of B cells with T cells andantigen presenting cells (APCs). Similar to CD19, CD22 is a B celllineage-restricted marker, expressed explicitly by B lymphoid cells fromthe pre-B to mature B cell stage. However, it is lost duringdifferentiation to plasma cells. CD22 is universally expressed in most Bcell malignancies, including acute lymphoblastic leukemia (ALL), chroniclymphocytic leukemia (CLL), and various subtypes of non-Hodgkin lymphoma(NHL) such as diffuse large B cell lymphoma. Targeting CD22 as anattractive therapeutic target for B cell malignancies has been confirmedby positive results in clinical trials of anti-CD22 monoclonalantibodies (e.g., epratuzumab) and immunotoxins (e.g., BL22, HA22). CD22has been shown to be expressed on ALL cells that lost CD19 expressionafter treatment with anti-CD19 CAR-T cells, making anti-CD22 CAR-T cellssuitable for combination and/or follow-on therapy of anti-CD19 CAR-Tcells.

However, although numerous clinical studies have demonstrated thepotential of adoptive transfer of CAR T cells for cancer therapy, theyhave also raised the risks associated with the cytokine-release syndrome(CRS) and the “on-target off-tumour” effect.

The mRNA nanoparticle delivery compositions provided herein are usefulto improve the safety and efficacy of CAR-T-cells. For example, the mRNAnanoparticle delivery systems of embodiments described herein may beused to recruit specific immune cells or modified subsets of immunecells such as CAR-T cells to the tumour microenvironment. Additionally,the mRNA nanoparticle delivery systems may be used to inhibit expressionof endogenous T cell receptors (TCRs) to avoid graft-versus-host diseaseand/or to selectively delete immune checkpoint genes in these cells tostrengthen their anti-cancer activity in the suppressive tumour milieu.(See e.g., Moffett, Coon, et al. (2017) “Hit-and-run programming oftherapeutic cytoreagents using mRNA nanocarriers.” NatureCommunications. 8:389.)

In some embodiments, the coding mRNA and the delivery particles are usedto attract CAR-T cells to a particular site in a subject. In someembodiments, the coding mRNA and the delivery particles are used toovercome insufficient migration of an immune cell to the tumourmicroenvironment. In response to specific chemokines, different immunecell subsets migrate into the tumour microenvironment and regulatetumour immune responses in a spatiotemporal manner. In addition,chemokines can directly target non-immune cells, including tumour cellsand vascular endothelial cells, in the tumour microenvironment, and theyhave been shown to regulate tumour cell proliferation, cancer stem-likecell properties, cancer invasiveness and metastasis. In someembodiments, the immune cell is a T cell, a natural killer (NK) cell, aB cell, an antigen-presenting cell (APC) such as a macrophage ordendritic cell, or any combination thereof.

In some embodiments, the coding mRNA and the delivery particles are usedto overcome insufficient migration of CAR T cells to the tumourmicroenvironment. In some embodiments, the delivery particlesspecifically target the tumour microenvironment, and the coding mRNAencodes a gene product that attracts or otherwise recruits CAR-T cellsto the tumour microenvironment. In some embodiments, the coding mRNAexpresses a chemokine. By way of non-limiting example, the coding mRNAcan encode a chemokine that attracts T-cells such as CCL2, CCL3, CCL4,CCL5, CCL20, CCL22, CCL28, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, XCL1,and any combination thereof. In situations where the reverse effect isdesired, such as in autoimmune disease, the coding mRNA can expressblockers, antagonists and/or inhibitors of the above-mentioned factors.

In some embodiments, the coding mRNA and delivery particles are used totransiently express the coding mRNA in the tumour microenvironment. Insome embodiments, the coding mRNA encodes a cytokine or other geneproduct involved in regulating the survival, proliferation, and/ordifferentiation of immune cells in the tumour response, such as, forexample, activated T cells and NK cells. By way of non-limiting example,the coding mRNA can encode for a cytokine such as IL-1, IL-2, IL-4,IL-6, IL-8, IL-10, IL-12, IL-17, IL-33, IL-35, TGF-beta, and anycombination thereof. Again, in situations where the reverse effect isdesired, such as in autoimmune disease, the coding mRNA can expressblockers, antagonists and/or inhibitors of the above-mentioned factors.

In some embodiments, of the invention, the coding mRNA and the deliveryparticle are used in conjunction with CAR-T or other adoptive celltherapy to provide transient expression of the coding mRNA.

In some embodiments, the mRNA nanoparticle delivery system delivers anmRNA that codes for a gene-editing agent to a target cell population. Insome embodiments, the mRNA codes for a sequence-specific nuclease thattargets a gene locus and disrupts expression of one or more endogenousgene produces in the target cell population. In some embodiments, themRNA codes for a sequence-specific nuclease that targets a T cellreceptor (TCR)-related gene locus, thereby disrupting expression of oneor more domains in the TCR.

In some embodiments, the mRNA nanoparticle delivery compositions may beused to deliver an mRNA that codes for one or more agents that programengineered T cells toward a desired phenotype. In some embodiments, themRNA nanoparticle delivery compositions may be used to induce markersand transcriptional patterns that are characteristic of a desired T cellphenotype. In some embodiments, the mRNA nanoparticle deliverycompositions may be used to promote development of CD26L+ central memoryT cells (Tcm), which have been shown to improve CAR-T treatment. (Seee.g., Moffett, Coon supra).

In some embodiments, the mRNA nanoparticle delivery compositions includea surface-anchored targeting domain that is specific for a T cellmarker, such as, for example, a surface antigen found on T cells. Insome embodiments, the surface-anchored targeting domain is specific foran antigen that selectively binds the nanoparticle to T-cells andinitiates receptor-induced endocytosis to internalize the mRNAnanoparticle delivery compositions. In some embodiments, thesurface-anchored targeting domain selectively binds CD3, CD8, or acombination thereof. In some embodiments, surface-anchored targetingdomain is or is derived from an antibody that selectively binds CD3,CD8, or a combination thereof.

MicroRNAs (miRNAs) are a class of noncoding RNAs each containing around20 to 25 nucleotides some of which are believed to be involved inpost-transcriptional regulation of gene expression by binding tocomplementary sequences in the 3′ untranslated regions (3′ UTR) oftarget mRNAs, leading to their silencing. These sequences are alsoreferred to herein as miRNA binding site, or miRNA binding sitesequences. Certain miRNAs are highly tissue-specific in theirexpression; for example, miR-122 and its variants are abundant in theliver and infrequently expressed in other tissues (Lagos-Quintana(2002), Current Biology, Vol. 12, April).

The miRNA system therefore provides a robust platform by which nucleicacids introduced into cells can be silenced in selected cell types in atarget tissue, and expressed in others. By including a binding site fora particular given miRNA sequence into an mRNA construct to beintroduced into target cells, particularly in or immediately 5′ or 3′ toa UTR, expression of certain introduced genes can be reduced orsubstantially eliminated in some cell types, while remaining in others(Brown and Naldini, Nature Reviews Genetics volume 10, pages 578-585(2009)). The use of the term ‘immediately’ is understood to besynonymous with terms such as ‘highly proximate to’ or ‘very close to’.When referring to 5′ or 3′ positioning relative to a UTR sequence itencompasses variants in which typically up to around twenty, suitablynot more than fifty, intervening nucleotide bases may be placed betweenthe miRNA binding sequence and the adjacent UTR. It is contemplated thatone, or a plurality, of such miRNA binding site sequences can beincluded in the mRNA construct. Where a plurality of miRNA binding sitesequences are present, this plurality may include for example greaterthan two, greater than three, typically greater than four miRNA bindingsite sequences. These miRNA binding site sequences may be arrangedsequentially, in tandem or at predetermined locations within, 3′ to, or5′ to a specified UTR within the mRNA constructs.

miR-122, despite its abundance in healthy non-diseased liver tissue, isreduced in the majority of liver cancers as well as in diseased cells(Braconi et al. 2011, Semin Oncol; 38(6): 752-763, Brown and NaldiniNature 2009; 10 578). By the above-mentioned method, it has been foundthat when the target tissue is the liver, translation of the introducedmRNA sequences can be facilitated in cancerous liver cells and reducedor substantially eliminated in transfected healthy cells, by includingmiRNA-122 binding sites (for example, SEQ ID NO: 1) in or adjacent totheir 3′ UTRs.

In the context of disease-specific expression of introducedpolynucleotides, binding sequences for any miRNA sequence which isdisrupted in a particular disease—that is, upregulated or downregulatedin diseased cells (such as tumour cells) in comparison to non-diseasedcells—is considered suitable for use in the invention. Table 2 discussesexamples of tumour-associated miRNA binding sequences of this kind whichmay be used in embodiments of the present invention. It will beappreciated, however, that the present invention is not limited only toinstances where a given miRNA or class of miRNAs is downregulated in afirst cell type versus a second cell type within a given organ or organsystem. On the contrary, it is merely required that there exists adifferential expression pattern of a regulatory miRNA between first andsecond cell types comprised within the organ or organ system. Thedifferential expression of the miRNA can be exploited using thecompositions and methods described herein to enable correspondingdifferential translation of protein products in those cells.

Examples of cancers where evidence has been found for similardifferential miRNA expression between healthy and cancer cells includebreast (Nygaard et al, BMC Med Genomics, 2009 Jun. 9; 2:35), ovarian(Wyman et al, PIS One, 2009; 4(4):e5311), prostate (Watahiki et al, PloSOne, 2011; 6(9):e24950), and cervical cancers (Lui et al. CancerResearch, 2007 Jul. 1; 67(13):6031-43). WO 2017/132552 A1 describes awide range of miRNAs with differing expression levels in various cancercells.

TABLE 2 Tissue/ Implicated cancer type miRNA Expression profileReference Liver miRNA-122 Reduced in cancer Braconi, 2011, cells Brown,2009 Liver miRNA-125 Reduced in hepato- Coppola N. carcinoma Oncotarget,2017. Vol 8 Brain miRNA-124a Reduced in Mazzacurati L. glioblastomaMoleculatherapy 23, 2015 Lung, breast Let-7 Reduced in Edge R E et al.cancer cells Mol Ther 2008; 16: 1437 Yu F. Cell 2007; 131 (6): 1109-23Pancreas miRNA-375 Reduced in Song S, Zhou J cancer cells et al. BiomedReports: 393-398, 2013

In the pancreas, miRNA-375 expression has been indicated to be high innormal pancreas cells but significantly lower in diseased and/orcancerous tissues (Song, Zhou et al. 2013). This expression has beenshown to relate to the stage of cancer, with expression further reducedwith more advanced cancer. It is thought that miRNA-375 is involved withthe regulation of glucose-induced biological responses in pancreatic0-cells, by targeting 3-phosphoinositide-dependent protein kinase-1(PDK1) mRNA and so affecting the PI 3-kinase/PKB cascade (El Ouaamari etal. Diabetes 57:2708-2717, 2008). An anti-proliferative effect ofmiRNA-375 is implicated by this putative mode of action, which mayexplain its downregulation in cancer cells.

The UTR of the mRNA sequences supplied by the present invention can beselected to have similarity, for example greater than 90% similarity, topart or all of a UTR sequence expressed in one of the cell types withinthe target organ. Particular cell types can have genes which are up- ordown-regulated in expression, and the UTR sequence can mediate thisregulation, for instance through encouraging the stability ordegradation of the relevant mRNA sequences.

As an example, UTRs associated with genes which are known to beupregulated in cancer cells may have one or more features, such as miRNAbinding site sequences, which encourage their stability and translationin these cancer cells. By incorporating similar sequences into suppliedmRNA sequences, stability and translation can be improved in cancerouscells but not non-cancerous or healthy cells.

It is also considered that the cancer to be treated by the invention maybe a secondary cancer in the target tissue, that is, a metastasis from acancer elsewhere than the target tissue. For example, a liver metastasismight originate from a cancer of the oesophageal, stomach, colon,rectum, breast, kidney, skin, pancreas or lung, and may beadenocarcinoma or another type of cancer. In these cases alternativemiRNA sequences may need to be selected in order to provide differentialexpression in healthy, non-cancerous and/or cancerous cells. Indeed,there may be an increased choice of candidate miRNA sequences in suchcases, due to the different tissue origin of metastasised cells.

In certain situations, it is possible that more than one candidate foran miRNA sequence which exhibits differential expression in differentcell types in a target tissue may exist. In such cases, it may beadvantageous that a plurality of miRNA binding site sequences areincluded in the mRNA construct, and that these sequences may besubstantially different sequences. However, it is also envisaged thateach of the plurality of miRNA binding site sequences may besubstantially the same sequence.

Combination Therapies

Oncolytic Viruses

As mentioned above, oncolytic viral therapy is the process of usingviruses to infect and kill cancer cells, sometimes by direct virallysis, but also including indirect killing by the stimulation of hostanti-tumoural responses. While oncolytic viruses are frequentlycharacterised by having increased activity in cancer cells compared withhealthy cells, off-target effects caused by damage to healthy cells havebeen documented (Russell et al. Nature Biotechnology, 2012).

In order to increase safety and decrease off-target effects, oncolyticviruses may be modified or selected to reduce their virulence, forexample by the deletion of virulence factors or genes involved infunctions such as intracellular immune system suppression and evasion,viral genome replication, and the takeover of host cell processes. Thehistorical production of safe forms of live viruses for use invaccination is another source of attenuated viruses. In other cases,particular mutations or even additional genes have been seen to enhanceoncolytic activity in particular oncolytic viruses. Non-exhaustiveexamples of the virulence genes commonly added, mutated or deleted inoncolytic viruses may be found in Table 3.

TABLE 3 Oncolytic virus Mutation Reference Vesicular G protein (Q242Rmutation) Brun et al 2010, Stomatitis M protein (L123W mutation) MolTher.; Virus, 18(8): 1440-1449. marabavirus Measles virus NIS gene -Human thyroidal Aref et al 2016, iodide symporter Viruses, 8, 294Newcastle disease Fusion protein (F) cleavage Vigil et al 2007 virussite Cancer Res; 67: (17). Parvovirus NS protein NS1 Marchini et al 2015Virology Journal 12:6 Herpes Simplex Viral ribonucleotide Liu et al(2003) Virus Type 1 reductase (ICP6) Gene Therapy (HSV-1) inactivation;volume 10, 292-303; serine/threonine-protein Goldsmith et al 1998 kinase(US3) J Exp Med. 187(3): inactivation; 341-348; ICP34.5 and ICP47inactivation (Neurovirulence and immune system evasion); UL43inactivation (Cell fusion) inactivation; UL49.5 inactivation (T-cellevasion) inactivation; UL55 and UL56 Adenovirus E1B-55, E3, E1apromoter, Baker et al 2018, E3 gp19 kD, E1A 924 bp), Cancers, 10, 201E1A, deletion in E3 and E4, E3 quaitotal deletion, chimeric ad3/Ad11pE2B region, E3-6.7K + gp19K E1A

The attenuation or modification of oncolytic viruses in this way canplay a role in the selectivity of oncolytic viruses to cancer cells:since the process of carcinogenesis often involves the inactivation ofgenes that play protective roles against both cancer (such as byregulating cell division or apoptosis), and viral infection, oncolyticviruses which are attenuated as described can retain their virulence incancer cells, due to the absence of the usual antiviral genes in thesecells. Therefore in healthy cells the attenuated virus cannot defendagainst the normal antiviral responses, and is eliminated, whereas incancer cells this response is absent, and the virus can lyse the cells.However, this approach is rarely completely effective, as firstlypartial inactivation of antiviral responses in cancer cells is morecommon than a complete lack of antiviral activity (Haralambieva et al,Mol. Ther., 2007), meaning that virulence can still be reduced in thesecells, and secondly infection of healthy cells can still occur.

Similarly, as viruses typically utilise the cellular machinery of thehost cell in order to replicate their genomes, but this machinery istypically downregulated in healthy, quiescent, non-replicating cellswhich are not replicating their own genomes, many viruses possess geneswhich reactivate or compensate for the host machinery. For example,ribonucleotide reductase enzymes are necessary for the production ofdeoxyribonucleotides from ribonucleotides; these enzymes are typicallydownregulated in quiescent host cells, and several viruses possess genesfor their own enzyme of this type, in order to have a source ofdeoxyribonucleotides. Since replicating cancer cells may have theseenzymes reactivated, an attenuated oncolytic virus with its ownribonucleotide reductase enzyme gene deleted can still replicate incancer cells. However, for reasons similar to the above, this approachmay not be completely effective, either in protecting healthy cells frominfection, or in restoring virulence in cancer cells. For example, notall cells in a tumour are replicating at any given time, and as suchsufficient deoxyribonucleotides may not be available for viralreplication in the majority of cancer cells.

Following the above, when a composition or method according to thepresent invention is used in conjunction with oncolytic viral therapy,the therapeutic enhancement factor provided by the constructs of theinvention may be a factor which increases the efficacy of the oncolyticvirus in cancer cells, for example enhancing replication of the virus,or the ability of the virus to lyse the cells in which it resides. Inparticular, where the oncolytic virus has been modified to attenuate itsfunction, for example by the deletion of one or more genes for virulencefactors, the therapeutic factor may replace the deleted gene with mRNAfor a gene product which is a copy of the viral gene product, or a geneproduct with substantial homology to the deleted gene, or whichotherwise compensates for the deletion of the gene. In such embodiments,by the differential expression in healthy and cancerous cells which ismade possible by the invention, the replacement gene product can beexpressed only in cancer cells, enhancing viral activity and lysis inthese cells, rather than in healthy cells, where expression of theprovided mRNA is inhibited by the presence of the miRNA binding sites.

By similar means, mRNA coding for factors which increase the resistanceof cells to oncolytic viruses can be expressed preferentially in healthycells, again promoting viral activity in cancerous cells compared tohealthy cells.

A benefit of this approach is that, unlike previous therapies usingoncolytic viruses, it does not rely on which cellular antiviral genesand processes may be inactivated due to carcinogenesis, nor on cellreplication processes which may be activated in some cancer cells butnot others. As a result, a greater scope of which virulence genes can bedeleted from oncolytic viruses is allowed. Thus, oncolytic viruses canbe modified to completely lack replicative ability in healthy cells,and, in cancer cells where the function of the deleted virulence genesare replaced by means of the invention, the virus can be restored tofull potency. As a result, side effects can be reduced, and efficacyincreased. Similarly, since the differential expression of the providedmRNA relies on miRNA expression differences between cancer and healthycells, virulence can be restored in all transfected cancer cells, andnot only those that, for example, are undergoing replication at time ofadministration.

In a particular embodiment, the oncolytic virus is HSV-1, part of theherpesvirus family. Attenuated versions of HSV may be engineered orselected to be deficient in ICP6, which encodes a viral ribonucleotidereductase (Aghi et al, Oncogene. 2008) and/or in US3, which encodes aserine/threonine-protein kinase, and plays several roles in the virus'lifecycle, including blocking host cell apoptosis (Kasuya et al, CancerGene Therapy, 2007).

Cytokines

It is contemplated that the compositions and methods as described hereinmay act to induce an immune response against disease. In particular,immune responses may be induced against cancer cells. The process ofcarcinogenesis frequently involves ways in which the cancer cellsattempt to evade the immune system, involving changes to the antigensproduced and displayed by these cells, In some embodiments, the mRNAprovided by the invention comprises at least one polynucleotide encodinga protein that is a bispecific T-cell engager (BiTE), ananti-immunosuppressive protein, or an immunogenic antigen. The term“anti-immunosuppressive protein” as used herein is a protein thatinhibits an immunosuppressive pathway.

The invention encompasses compositions supplying mRNA coding for ananti-immunosuppressive protein that is an anti-regulatory T-cell (Treg)protein or an anti-myeloid-derived suppressor cell (MDSC) protein. Insome embodiments, the anti-immunosuppressive protein is a VHH-derivedblocker or a VHH-derived BiTE.

The term “immunogenic antigen” as used herein refers to a protein thatincreases an inflammatory or immunogenic immune response. In particularembodiments, the anti-immunosuppressive and immunogenic antigens inducean anti-tumour immune response. Examples of such proteins includeantibody or antigen binding fragments thereof that bind to and inhibitimmune checkpoint receptors (e.g. CTLA4, LAG3, PD1, PDL1, and others),proinflammatory cytokines (e.g., IFNγ, IFNα, IPNβ, TNFα, IL-12, IL-2,IL-6, IL-8, GM-CSF, and others), or proteins that binding to andactivate an activating receptor (e.g., FcγRI, FcγIIa, FcγIIa,costimulatory receptors, and others). In particular embodiments, theprotein is selected from EpCAM, folate, IFNβ, anti-CTLA-4, anti-PD1,A2A, anti-FGF2, anti-FGFR/FGFR2b, anti-SEMA4D, CCL5, CD137, CD200, CD38,CD44, CSF-1R, CXCL10, CXCL13, endothelin B Receptor, IL-12, IL-15, IL-2,IL-21, IL-35, ISRE7, LFA-1, NG2 (also known as SPEG4), SMADs, STING,TGFβ, and VCAM1.

The invention encompasses compositions supplying mRNA coding forfunctional macromolecules to targeted cell populations used incell-based therapies. In some embodiments, the targeted cell populationis a genetically engineered T cell population. In some embodiments, thetargeted cell population is a population of chimeric antigen receptor Tcells (CAR-T cells).

The coding mRNA and the delivery particles may be used to attract apopulation of immune cells or a combination of immune cell populationsto a particular site in a subject. In some embodiments, the coding mRNAand the delivery particles are used to attract immune cells to thetumour microenvironment. In some embodiments, the coding mRNA and thedelivery particles are used to overcome insufficient migration of animmune cell to the tumour microenvironment. In some embodiments, theimmune cell is a T cell, a natural killer (NK) cell, a B cell, anantigen-presenting cell (APC) such as a macrophage or dendritic cell, orany combination thereof. In some embodiments, the coding mRNA and thedelivery particles are used to attract CAR-T cells to the tumourmicroenvironment.

The coding mRNA and the delivery particles may be used to overcomeinsufficient migration of CAR T cells to the tumour microenvironment. Insome embodiments, the delivery particles specifically target the tumourmicroenvironment, and the coding mRNA encodes a gene product thatattracts or otherwise recruits CAR-T cells to the tumourmicroenvironment. In some embodiments, the coding mRNA expresses achemokine. By way of non-limiting example, the coding mRNA can encode achemokine that attracts T-cells such as CCL2, CCL3, CCL4, CCL5, CCL20,CCL22, CCL28, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, XCL1, and anycombination thereof. In situations where the reverse effect is desired,such as in autoimmune disease, the coding mRNA can express blockers,antagonists and/or inhibitors of the above-mentioned factors.

The coding mRNA and the delivery particles may be used to transientlyexpress the coding mRNA in the tumour microenvironment. In someembodiments, the coding mRNA encodes a cytokine or other gene productinvolved in regulating the survival, proliferation, and/ordifferentiation of immune cells in the tumour response, such as, forexample, activated T cells and NK cells. By way of non-limiting example,the coding mRNA can encode for a cytokine such as IL-1, IL-2, IL-4,IL-6, IL-8, IL-10, IL-12, IL-17, IL-33, IL-35, TGF-beta, and anycombination thereof. Again, in situations where the reverse effect isdesired, such as in autoimmune disease, the coding mRNA can expressblockers, antagonists and/or inhibitors of the above-mentioned factors.

The compositions supplying mRNA may be designed to target particularcell subtypes and, upon binding to them, stimulate receptor-mediatedendocytosis, thereby introducing the synthetic mRNA they carry to thecells, which can now express the synthetic mRNA. Because nucleartransport and transcription of the transgene are not required, thisprocess is fast and efficient.

In some embodiments, the mRNA nanoparticle delivery system delivers anmRNA that codes for a gene-editing agent to a target cell population. Insome embodiments, the mRNA codes for a sequence-specific nuclease thattargets a gene locus and disrupts expression of one or more endogenousgene produces in the target cell population. In some embodiments, themRNA codes for a sequence-specific nuclease that targets a T cellreceptor (TCR)-related gene locus, thereby disrupting expression of oneor more domains in the TCR.

In some embodiments, the mRNA nanoparticle delivery compositions may beused to deliver an mRNA that codes for one or more agents that programengineered T cells toward a desired phenotype. In some embodiments, themRNA nanoparticle delivery compositions may be used to induce markersand transcriptional patterns that are characteristic of a desired T cellphenotype. In some embodiments, the mRNA nanoparticle deliverycompositions may be used to promote development of CD26L+ central memoryT cells (Tcm), which have been shown to improve CAR-T treatment. (Seee.g., Moffett, Coon supra). In some embodiments, compositions supplymRNA encoding one or more transcription factors to control celldifferentiation in a target cell population. In some embodiments, thetranscription factor is Foxol, which controls developmenteffector-to-memory transition in CD8 T-cells.

In some embodiments, the mRNA nanoparticle delivery compositions includea surface-anchored targeting domain that is specific for a T cellmarker, such as, for example, a surface antigen found on T cells. Insome embodiments, the surface-anchored targeting domain is specific foran antigen that selectively binds the nanoparticle to T-cells andinitiates receptor-induced endocytosis to internalize the mRNAnanoparticle delivery compositions. In some embodiments, thesurface-anchored targeting domain selectively binds CD3, CD8, or acombination thereof. In some embodiments, surface-anchored targetingdomain is or is derived from an antibody that selectively binds CD3,CD8, or a combination thereof.

By means of the invention, differential expression of theabove-mentioned gene products can be achieved in different cell types,for example, in healthy cells, non-diseased, diseased and cancer cells.By this method, an immune response can be triggered targeted towardsdiseased cells while sparing the non-diseased or healthy cells.

The introduction of coding nucleotides sequences into a target cell mostoften requires the use of a delivery agent to transfer the desiredsubstance from the extracellular space to the intracellular environment.Frequently, such delivery agents are in the form of delivery particles,which may undergo phagocytosis and/or fuse with a target cell. Deliveryparticles may contain the desired substance by encapsulation or bycomprising the substance within a matrix or structure.

The delivery particles of the present invention may be targeted to thecells of the target tissue. This targeting may be mediated by atargeting agent on the surface of the delivery particles, which may be aprotein, peptide, carbohydrate, glycoprotein, lipid, small molecule,nucleic acid, etc. The targeting agent may be used to target specificcells or tissues or may be used to promote endocytosis or phagocytosisof the particle. Examples of targeting agents include, but are notlimited to, antibodies, fragments of antibodies, low-densitylipoproteins (LDLs), transferrin, asialycoproteins, gp120 envelopeprotein of the human immunodeficiency vims (HIV), carbohydrates,receptor ligands, sialic acid, aptamers etc.

Typically, the delivery particles comprise aminoalcohol lipidoids. Thesecompounds may be used in the formation of particles includingnanoparticles, liposomes and micelles, which are particularly suitablefor the delivery of nucleic acids. An illustrative example for theproduction of nanoformulations comprising particles according to someembodiments of the invention may be found in the Examples.

When administered to a subject, a therapeutic component is suitablyadministered as part of a composition that comprises a pharmaceuticallyacceptable vehicle. Acceptable pharmaceutical vehicles can be liquids,such as water and oils, including those of petroleum, animal, vegetableor synthetic origin, such as peanut oil, soybean oil, mineral oil,sesame oil and the like. The pharmaceutical vehicles can be saline, gumacacia, gelatin, starch paste, talc, keratin, colloidal silica, urea,and the like. In addition, auxiliary, stabilising, thickening,lubricating and colouring agents may be used. When administered to asubject, the pharmaceutically acceptable vehicles are preferablysterile. Water is a suitable vehicle when the compound of the inventionis administered intravenously. Saline solutions and aqueous dextrose andglycerol solutions can also be employed as liquid vehicles, particularlyfor injectable solutions. Suitable pharmaceutical vehicles also includeexcipients such as starch, glucose, lactose, sucrose, gelatin, malt,rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate,talc, sodium chloride, dried skimmed milk, glycerol, propylene, glycol,water, ethanol and the like. Pharmaceutical compositions, if desired,can also contain minor amounts of wetting or emulsifying agents, orbuffering agents.

The medicaments and pharmaceutical compositions of the invention cantake the form of liquids, solutions, suspensions, gels, modified-releaseformulations (such as slow or sustained-release), emulsions, capsules(for example, capsules containing liquids or gels), liposomes,microparticles, nanoparticles or any other suitable formulations knownin the art. Other examples of suitable pharmaceutical vehicles aredescribed in Remington's Pharmaceutical Sciences, Alfonso R. Gennaroed., Mack Publishing Co. Easton, Pa., 19th ed., 1995, see for examplepages 1447-1676.

For any compound or composition described herein, the therapeuticallyeffective amount can be initially determined from in vitro cell cultureassays. Target concentrations will be those concentrations of activecomponent(s) that are capable of achieving the methods described herein,as measured using the methods described herein or known in the art.

As is well known in the art, therapeutically effective amounts for usein human subjects can also be determined from animal models. Forexample, a dose for humans can be formulated to achieve a concentrationthat has been found to be effective in animals. The dosage in humans canbe adjusted by monitoring compounds effectiveness and adjusting thedosage upwards or downwards, as described above. Adjusting the dose toachieve maximal efficacy in humans based on the methods described aboveand other methods is well within the capabilities of the ordinarilyskilled artisan.

It is contemplated that embodiments of the invention may includecompositions formulated for use in medicine. As such, the composition ofthe invention may be suspended in a biocompatible solution to form acomposition that can be targeted to a location on a cell, within atissue or within the body of a patient or animal (i.e. the compositioncan be used in vitro, ex vivo or in vivo). Suitably, the biocompatiblesolution may be phosphate buffered saline or any other pharmaceuticallyacceptable carrier solution. One or more additional pharmaceuticallyacceptable carriers (such as diluents, adjuvants, excipients orvehicles) may be combined with the composition of the invention in apharmaceutical composition. Suitable pharmaceutical carriers aredescribed in ‘Remington's Pharmaceutical Sciences’ by E. W. Martin.Pharmaceutical formulations and compositions of the invention areformulated to conform to regulatory standards and can be administeredorally, intravenously, topically, intratumorally, or subcutaneously, orvia other standard routes. Administration can be systemic or local orintranasal or intrathecal.

Further intended are embodiments wherein the composition of someembodiments of the invention is administered separately to or incombination with alternative antitumoral or otherwise anti-cancertherapeutic components. These components can include oncolytic viruses,small molecule drugs, chemotherapeutics, radiotherapeutics orbiologicals. The components may be administered concurrently with thecomposition of the invention, and may be comprised within the deliveryparticles, or may be administered separately, before or afteradministration of the composition of the invention, by any meanssuitable.

It is also contemplated that the composition of some embodiments of theinvention may be used in in vitro and/or ex vivo methods, for example ina laboratory setting. An example of an in vivo method is wherein acomposition comprising a delivery particle and an mRNA sequence asdescribed herein is administered to target in vitro cells, and the miRNAbinding site sequences comprised in the mRNA sequence allow fordifferential expression of the coding sequence of the mRNA in differentcell types within the target in vitro cells. Similarly, a method iscontemplated wherein a composition comprising a delivery particle and anmRNA sequence as described herein is administered to a target ex vivosample taken from an animal, and the miRNA binding site sequencescomprised in the mRNA sequence allow for differential expression of thecoding sequence of the mRNA in different cell types within the targetsample.

The device of the invention is exemplified by, but in no way limited to,the following Examples.

EXAMPLES

General Protocols

Cell Lines

Human liver hepatocarcinoma (HCC) HepG2 (ATCC® HB-8065™) and Hep3B(ATCC® HB-8064™) cell were purchased from ATCC. Cells were cultured inEagle's Minimum Essential Medium (EMEM) (Cellgro, USA), 10% FBS(HyClone, USA), streptomycin (100 μg/mL) and penicillin (100 U/mL⁻¹)(Cellgro) as monolayers at 37° C. in an atmosphere of 5% CO₂. HepG2cells were grown on collagen (Gibco, USA) coated plates at a collagenconcentration of 5 μg/cm².

HMCPP5 (pooled plateable human hepatocytes; a mixture of plateableprimary hepatocytes produced by combining cells from 5 individualdonors) were purchased from ThermoFisher Scientific, USA. Cells wereplated in Williams E Medium (WEM), supplemented with 5% FBS, 1 μMDexamethasone, and Cocktail A (Penicillin/Streptomycin, HumanRecombinant Insulin, GlutaMax, and HEPES, pH 7.4). 24 hours afterplating, the WEM/Cocktail A medium was changed to maintenance/incubationmedium WEM supplemented with 0.1 μM Dexamethasone and Cocktail B(Penicillin/Streptomycin, ITS (Human Recombinant Insulin, Humantransferrin, selenous acid, BSA, linoleic acid), GlutaMax, and HEPES, pH7.4) as monolayers at 37° C. in an atmosphere of 5% CO₂. During allexperiments, cells were cultured in WEM/Cocktail B medium with theexception of during transfection with nanoformulated mRNA. WEM/CocktailB medium was exchanged for fresh every 3 days. HMCPP5 cell growth oncollagen (Gibco) coated plates at protein concentration 5 μg/cm².

Aml12 (mouse healthy hepatocytes) were purchased from ATCC, USA. Cellswere seeded into a 12-well plate at a density of 1×10⁵/well.

Vector Constructs

Constructing of pMRNA-CTx-mRNA Template

Plasmid pMRNA-CTx-mRNA template forming matrices for in vitro synthesisof all mRNA used in the experiments were constructed according tocommercially available mRNAExpress™ mRNA Synthesis Kit (SBI, USA). Allplasmids were propagated in E. coli (Invitrogen, USA) and purified usingQiagen Mini or Maxi Kit (Qiagen, USA). Restriction maps of all plasmidswere generated using pDRAW32 software (www.acaclone.com).

Cloning Method 1—Restriction Endonuclease

As shown in FIG. 2, the sequence of one or more genes, flanked withKozak sequence for optimal translation directly before the ATG codon atthe 5′-end or a stop codon (TAA, TAG, TGA) at the 3′-end of gene, weresynthesised by company GeneArt (without codon optimisation) anddelivered as plasmid DNA (referred to as DNA plasmids or vectors), shownin FIG. 2 as pMA-T-CTx-Gene. Proprietary 5′ and 3′ UTR regions flankingthe coding sequence were included in all synthesised sequences (notshown in the appended sequences). The 5′ UTR is synthetic, and containsthe Kozak sequence, and the 3′ UTR is based on a mouse alpha globin UTRand also comprises a poly A tail of 120 bases. To generate the synthesisvector shown in FIG. 2 as pMRNA-CTx-mRNA, a nucleotide fragmentcontaining the gene or genes was cut out from the DNA plasmid usingrestriction endonucleases, here EcoRI and NheI, and subcloned intoEcoRI/NheI restriction sites in the pMRNA template plasmid, thistemplate plasmid comprising T7 promoter recognized by T7 RNA polymerase,5′- and 3′-UTRs, and a polyA sequence.

Cloning Method 2—Cold Fusion

The sequence of one or more genes, flanked as in Cloning Method 1 with aKozak sequence and a stop codon (TAA, TAG, TGA), was synthesised bycompany GeneArt (without codon optimisation) and delivered as a plasmidDNA pMAT-CTx-Gene, with the backbone of this plasmid the same asdescribed above. To construct the pMRNA-CTx-mRNA template vector, theCold Fusion cloning kit (SBI, USA) was deployed. Briefly, the genesequence from the DNA plasmid was amplified by PCR with specificprimers, the primers adding an extension of 14 bases of homology to eachend of the gene sequence. These 14 bases were designed to be homologousto the ends of the linearised vector produced by digestion of thetemplate plasmid with a restriction endonuclease cutting in themulticloning site located between the 5′ and 3′ UTRs. To produce thesynthesis vector, the predicted PCR product was purified by PCRpurification kit (Qiagen, USA) and incorporated to the pMRNA templateplasmid following a Cold Fusion reaction (homology recombination)according to the manufacturer's protocol.

Construction of a Template Containing miRNA Binding Site Sequences

For the production of mRNA sequences comprising miRNA binding sitesequences, for which examples using miR-122 are shown in FIG. 3,exemplary methods of creating three variants are discussed below. Invariant 1, two copies of the miRNA binding site sequence are includedbetween the stop codon and the +1 position of the 3′ UTR. In variant 2,two copies of the miRNA binding site sequence are included at thebeginning or 5′ end of the 3′ UTR, and in variant 3, two copies of themiRNA binding site sequence are included at the end or 3′ end of the 3′UTR.

FIG. 4 shows examples of synthesis vectors comprising these threevariants, using as an example Protein B, a gene of approximately 1400base pairs.

Variant 1

As shown in FIG. 5, the sequence of one or more genes, flanked as in theabove methods with a Kozak sequence and a stop codon (TAA, TAG, TGA),and additionally comprising two copies of a miRNA binding site sequencefollowing the stop codon, was synthesised by company GeneArt (withoutcodon optimisation) and delivered as a plasmid (here illustrated againwith Protein A) DNA pMAT-CTx-Gene, with the backbone of this plasmid thesame as described above. This sequence was then cloned into the templateplasmid to create a synthesis vector by either of the methods describedabove.

Variants 2 and 3

The sequence of one or more genes, flanked as in the above methods witha Kozak sequence and a stop codon (TAA, TAG, TGA), and additionallycomprising a 3′ UTR, including two copies of a miRNA binding sitesequence either at the beginning/5′ end of this region (variant 2, asshown in FIG. 6) or at the end/3′end of this region (variant 3, as shownin FIG. 7), was synthesised by company GeneArt (without codonoptimisation) and delivered as a plasmid DNA pMAT-CTx-Gene, with thebackbone of this plasmid the same as described above. This sequence wasthen cloned into a template plasmid to create a synthesis vector byeither of the methods described above, modified in that restrictionenzymes were chosen (here EcoRI and Notl) to remove the 3′ UTR from thetemplate vector, such that the 3′ UTR from the supplied DNA sequencewould be present in the final synthesis vector, as this contained themiRNA binding site sequences.

In Vitro Transcription (IVT) of mRNA with in Vitro mRNA Synthesis

To perform IVT of mRNA with or without miRNA-modified 3′ UTRs thecommercially available mRNAExpress™ mRNA Synthesis Kit was used. DNAtemplates for IVT vectors were constructed as described in the protocolsas set forth above. The procedure of in vitro mRNA synthesis wasperformed according to the manufacturer's protocol. Briefly, a polyAtail was added to the DNA sequence using a PCR reaction with specific 5′and 3′ primers (provided with the kit). During in vitro transcription,the synthesised mRNA on DNA template was capped with anti-reverse capanalog (ARCA)-modified nucleotides (5-Methylcytidine-5′-Triphosphate).Cap analog, pseudouridine-5′-triphosphate and poly-A tail wereincorporated in the in vitro transcribed mRNAs to enhance stability andto reduce the immune response of host cells.

Synthesis of DMP^(CTx) and Formulation of mRNA

The delivery and modulation platform of Combined Therapeutics(DMP^(CTx)) formulation is a multi-component nanoparticle of ionizablelipid-like material C12-200, phospholipid DOPE, cholesterol andlipid-anchored polyethylene glycol C14-PEG-DSPE2000 mixture. Thisparticular composition of DMP^(CTx) and specific weight ratio (10:1) ofC12-200:mRNA and molar [%] composition of lipid-like material,phospholipid, cholesterol and PEG (Table 4) was optimized and hasrevealed high efficiency of the formulation in vivo (Kauffman K. J.,Nano Letter. 2015, 15, 7300-7306). The chemical structures of theseexemplary components are shown in FIG. 8.

To synthesize DMP^(CTx) an ethanolic solution (Mix A) of C12-200 (WuXi,China), as shown in FIG. 9A, phospholipid DOPE(1,2-dioleyl-snglycero-3-phosphoethanolamine) (Avanti Polar Lipids,Alabaster, Ala., USA), cholesterol (Sigma, USA) and C14-PEG-DSPE2000(Avanti Polar Lipids, Alabaster, Ala., USA) and an aqueous bufferedsolution of mRNA (Mix B) in (10 mM citrate, pH 4.5) was prepared. Bothethanolic Mix A and aqueous Mix B at ratio 3:1 were mixed/combined usingsyringe pumps and microfluidic chip device (Chen D, at al J. Am. Chem.Soc. 2012, 134 (16), 6948-6951). Alcoholic solution of nanoformulatedmRNA from microfluidic chip was collected to the 1.5 mL tubes.

TABLE 4 Formulation Compound Weight Ratio Molar Composition [%]C12-200:mRNA 10:1 n/a C12:200 n/a 35 DOPE 16 Cholesterol 46.5C14-PEG-DSPE200 2.5

To remove alcohol after formulation the DMP^(CTx)-mRNA mixture wastransferred to the Slide-A-Lyzer® Dialysis Cassette G2 and dialyzed inPBS in room temperature on the magnetic stirrer for 4 hours.Subsequently, formulated mRNA using syringe with 18 gauge, 1-inchbeveled needles was transferred to new 1.5 mL tubes and ready forcharacterization.

To calculate efficacy of mRNA encapsulation RiboGreen RNA assay(Invitrogen) was used according manufacture protocol. Polydispersity(PDI) and size of lipid nanoparticles was measured using dynamic lightscattering (ZetaPALS, Brookhaven, Instruments). The surface charge ofDMP^(CTx) (Zeta potential) was measured using the same instrument.Solutions of mRNA sequences with and without two copies of an miR-122sequence connected by a linker (SEQ ID NO: 2) and inserted after thestop codon of the coding mRNA sequence were prepared from a 1.05 and 1mg/ml stock, respectively. Examples of parameters after encapsulation ofmRNA sequences comprising the mCherry (mCh) sequence, the sequence ofprotein A, a human protein of approximately 25 kDa, are shown in Table5, including the size, encapsulation efficiency and polydispersity ofthe delivery and modulation platform of Combined Therapeutics(DMP^(CTx)). An illustrative diagram of a delivery particle according toDMP^(CTx) may be seen in FIG. 9B.

TABLE 5 Formula- Conc Encapsulation Size Poly- mRNA tion (ug/mL)efficacy (%) (nm) dispersity Protein A - 022 C12-200 202 78 93 0.12US3 - 052 172 78 93 0.12 mCherry - 062 120 76 96 0.12

Differential Expression of Delivered mRNA Constructs in Vitro

To investigate the potential of the present invention to successfullytransfect target cells with construct mRNA and subsequently drivedifferential expression in different cell types, the DMP^(CTx) mRNAplatform, modified with miRNA-122 binding sites, was used in a model ofliver hepatocarcinoma.

Transfection of Cell Lines

Fluorescence Imaging and Quantification

Single transfections of the human liver hepatocarcinoma cell lines HepG2and Hep3B were performed as follows: one day prior to transfection,HepG2 and Hep3B cells were seeded separately into a 12-well plate at adensity of 2.7×10⁵/well, and 2×10⁵/well (EMEM/10% FCS), respectively.The next day, cells were transfected either with a vehicle control ofPBS alone, with 0.5 μg/well of mRNA-mCherry-DMP^(CTx), or with 0.5μg/well of mRNA-mCherry-122-DMP^(CTx) (the sequence comprising SEQ IDNO: 3). The transfection was carried out by direct addition ofmRNA-DMP^(CTx) to the cultured medium in the well, with gentle mixtureof the cultured cells as needed.

Single transfections of HMCPP5 (pooled plateable human hepatocytes),were performed as follows: one day prior to transfection HMCPP5 cellswere seeded into a 12-well plate at a density of 2.5×10⁵/well(WEM/Cocktail B). The next day, cells were transfected either with avehicle control of DMP^(CTx) (PBS), with 0.5 μg/well ofmRNA-mCherry-DMP^(CTx), or with 0.5 μg/well ofmRNA-mCherry-122-DMP^(CTx). The transfection was carried out by thedirect addition of mRNA-DMP^(CTx) to the cultured medium in the well,with gentle mixture of the cultured cells as needed. During transfectionof HMCPP5 the WEM/Cocktail B medium was supplemented with 5% FBS.Transfection was carried out in the manner described for liver cancercells, above. 24 hours after transfection, the medium was again changedto WEM/Cocktail B.

To evaluate constitutive activity and expression of miRNA-122 in healthyhuman hepatocytes, multiple transfections of HMCPP5 cells were performedas follows: the HMCPP5 cells were seeded and cultured as above, and weretransfected with mRNA-mCherry-DMP^(CTx), or mRNA-mCherry-122-DMP^(CTx),three times (MPT) in total, with an interval of 48 hrs between eachtransfection. Transfection was carried out in the same manner describedfor single transfections of HMCPP5, as described above.

Single transfections of mouse healthy hepatocytes (Aml12, ATCC, USA)were performed as follows: one day prior to transfection Aml12 cellswere seeded into a 12-well plate at a density of 1×10⁵/well. The nextday, cells were transfected either with a vehicle control of DMP^(CTx)(PBS), with 0.5 μg/well of mRNA-mCherry-DMP^(CTx), or with 0.5 μg/wellof mRNA-mCherry-122-DMP^(CTx). The transfection was carried out by thedirect addition of mRNA-DMP^(CTx) to the cultured medium in the well,with gentle mixture of the cultured cells as needed.

Following transfection, mCherry expression in the above cell lines wasdetected using a fluorescence imaging system (application from EVOS® FLImaging Systems). Pictures showing mCherry fluorescence were taken 16,24, 48, 72, 96 and 144 hours after transfection.

Quantification of the mCherry fluorescence signal was performed usingImageJ software (NIH, USA) from 3 randomized fields on culture plates(mRNA-mCherry, mRNA-mCherry-122). FIGS. 10B, 11 and 12B show the resultsof such quantifications. The pixel count of mCherry transfected wellswere set at 100% (mCherry fluorescence). Statistical significance wasdetermined using the Student t-test. Results are shown as means±SD.Significant difference was defined with p value <0.05. Asterisksindicate a statistically significant difference between mCherryfluorescence in cells transfected with mRNA-mCherry, compared to cellstransfected with mRNA-mCherry-122 (****, p<0.0001, ***p<0.001, **p<0.01,*p<0.05).

Example 1: Tumor-Specific Gene Expression by miRNA-122 Regulation

miRNA-122 is an abundant, liver-specific miRNA, the expression of whichis significantly decreased in human primary hepatocarcinoma (HCC) andHCC derived cell lines such as Hep3B and HepG2. The objective of thisstudy was to demonstrate that modification of the 3′-untranslated region(UTR) of an mRNA sequence by the insertion of miRNA-122 targetedsequences (for example, SEQ ID NO: 2, as illustrated in variant 1, topof FIG. 3) may result in translational repression and/or deadenylationfollowed by decapping of exogenous mRNA in normal hepatocytes, but notin tested HCC cell lines.

To examine endogenous miRNA-122 activity in healthy hepatocytes, HMCPP5cells (pooled plateable human hepatocytes, which are a mixture ofplateable primary hepatocytes produced by combining cells from 5individual donors) were transfected with mRNA-mCherry ormRNA-mCherry-122 prepared according to the above general protocols,using mCherry (red fluorescent protein) as the introduced gene ofinterest and followed mCherry (red fluorescent protein) expression overtime. As illustrated in FIG. 10A, mCherry (mCh) expression was analyzedby fluorescence microscopy 48 hours post-transfection. During theentirety of the post-transfection time, mCherry expression was observedin HMCPP5 cells transfected with mRNA-mCherry (that is, without the3′UTR modification to introduce an miR-122 sequence), indicatingsuccessful transfection and translation. In contrast, in healthyhepatocytes, which are known to be miRNA-122 positive, the expression ofmRNA-mCherry-122 was downregulated to practically undetectable levelscomparable to those seen in control untransfected cells, even 3 daysafter transfection. This indicated that the presence of an miRNA-122targeted sequence in the mRNA-mCherry-122 inserted in 3′ UTR (Variant 1)prevents translation of the mRNA, most likely due to translationalrepression in recipient cells.

Quantification of the fluorescence signal exhibited by these cellsconfirmed the above. As shown in FIG. 10B, fluorescence intensity wasdrastically reduced in healthy cells transfected with mRNA-mCherry-122,compared with those transfected with mRNA-mCherry.

The result obtained in the experiment above showed that nativeexpression of miRNA-122 and colocalisation with an miRNA-122 targetedsequence (Variant 1) could efficiently regulate protein expression inhealthy hepatocytes thereby significantly increasing tumor-specific geneexpression. In the following experiment, the constitutive expression andactivity of miRNA-122 in HMCPP5 cells was evaluated. The HMCPP5 cellswere transfected with mRNA-mCherry or mRNA-mCherry-122 three times intotal, with an interval of 48 hr each time. Six days after the firsttransfection (that is, 48 hours after the last transfection) expressionof mCherry was determined by fluorescent microscopy. As before, whilecells transfected with the mRNA-mCherry construct exhibited clear redfluorescence, those transfected with the mRNA-mCherry-122 construct didnot. In FIG. 11, comparisons between cells transfected withmRNA-mCherry-122, and those transfected with mRNA-mCherry are shown overa five-day period after final transfection, both for singly (ST) andmultiply-transfected cells (MPT). Multiply-transfected cells can be seento exhibit the same drastic reduction in fluorescence intensity whentransfected with mRNA-mCherry-122 as singly-transfected cells, with theeffect lasting longer following multiple transfections. As would beexpected, this indicates that the differential expression effect drivenby the miRNA control mechanism is robust to repeated transfectionevents, and that the amount of miRNA-122 available within the cells todrive this mechanism is not exhausted in these timeframes.

To examine the effect of endogenous miRNA-122 activity using the humanliver hepatocarcinoma Hep3B and hepatoblastoma HepG2 cell lines, anexperiment similar to the above was carried out. Cells were transfectedwith the mRNA sequence mRNA-mCherry, mRNA-mCherry-122 (Variant 1), orunderwent a control transfection. As previously, after 48 hours,fluorescence microscopy was used to determine the expression of mCherryin the transfected Hep3B and HepG2 cells, as shown in FIG. 10A. In Hep3Bcells (FIG. 10A, middle column), mCherry fluorescence was clearly seenboth in the mRNA-mCherry and the mRNA-mCherry-122 transfected lines,indicating that the miRNA-122 mediated translation repression is notactive in these cells. In HepG2 cells, mCherry fluorescence was clearlyseen in the mRNA-mCherry transfected line, but while some fluorescencewas evident in the mRNA-mCherry-122 transfected cells, it appeared to beonly partially reduced and significantly greater than that seen innormal hepatocytes. Further evidence of this is shown in FIG. 10B, wherequantification of mCherry fluorescence in mRNA-mCherry-122 transfectedlines indicates that no reduction of fluorescence is shown in Hep3Bcells, but a reduction of around 50% is seen in the HepG2 cells.

The partial downregulation seen in HepG2 cells further implicatesmiRNA-122 mediated effects on translation, as cells from this line havebeen shown to retain residual miRNA-122 activity (Demonstration of thePresence of the “Deleted” MIR122 Gene in HepG2 Cells, PLoS One. 2015;10(3)).

miRNA-122 is strongly conserved between vertebrate species, and, as inthe human, a reduced level of miRNA-122 is associated withhepatocellular carcinoma in mouse (Kutay et al, 2006). The endogenouseffect of miRNA-122 activity was therefore examined, using the mousehealthy hepatocyte cell line Aml12.

Healthy mice hepatocytes were also transfected with the mRNA-Cherrysequences previously described, i.e. mRNA-mCherry or mRNA-mCherry-122,encapsulated in DMP^(CTx). A similar impact of the insertion ofmiRNA-122 binding site sequence on the mCherry fluorescence was observedat 24 and 72 hours after transfection.

As shown in FIG. 12A, fluorescence was observed after transfection withmRNA-mCherry. A marked reduction in fluorescence was shown aftertransfection with mRNA-mCherry-122, although some signal could still beseen.

Quantification of mCherry fluorescence 24 and 72 hr after transfectionwas performed from 3 randomised fields on culture plates from eachtreatment group (FIG. 12B) showed that when transfected withmRNA-mCherry-122, more than 70% translation repression was observed.

As a preliminary conclusion, the above Example shows that the doubletargeting features of the nanoparticle delivery system, and theinclusion of miRNA-122 target sequence in the mRNA construct aresufficient to obtain quite significant differential expression of aprotein product in hepatocarcinoma and hepatoblastoma cells compared tohealthy hepatocytes. The observation of differential expression wasevident in both human and mouse cell lines.

Example 2: Protein Expression Level after Tumor-Specific Gene Expression

In another experiment, Western blotting was employed to determineprotein expression level ultimately exhibited after transfection asfollows.

Transfection of Cell Lines and Immunoblot—Protein A

To evaluate tumor specific expression level of an exemplary 25 kDa humanprotein (denoted ‘protein A’) both liver cancer cells (HepG2 and Hep3B)and healthy hepatocytes (HMCPP5) were seeded into 12-well plates andtransfected with 0.5 μg/well of nanoformulated mRNA expressing humanprotein A, 25 kDa (mRNA-A-DMP^(CTx)) or mRNA expressing human protein A(a human protein of approximately 25 kDa) comprising two miRNA122binding sequences in the 3′ UTR (SEQ ID NO: 2), Variant 1(mRNA-A-miRNA122-DMP^(CTx)), as described above in Example 1 for mCherrytransfection. 24 hours after transfection, immunoblot was performedfollowing total protein extraction.

For the immunobiot, culture media was removed, cells were washed withcold PBS (Celigro) and cell pellets lysed in RIPA(radioimmunoprecipitation assay) buffer (Boston Bioproducts) with acocktail of protease inhibitors (Sigma). Protein concentration wasdetermined by colorimetric Bradford assay. A total of 10 mg of proteinwas separated by Novex™ 4-12% mini gels (ThermoFisher Scientific) andtransferred onto PVDF (polyvinylidene difluoride) membranes byelectroblotting (iBlotO 2 Gel Transfer Device, Invitrogen). Afterblocking with 5% nonfat dry milk in TBS-Tween 20 (Boston Bioproducts),membranes were incubated at 4° C. overnight with anti-protein Aantibodies (1:2000, Abcam), or β-actin (Cell Signaling), followed byincubation with appropriate HRP (horseradish peroxidase)-conjugated goatanti-rabbit secondary antibodies (1:10000; Abcam) for 1 hour at roomtemperature. Protein-antibody complexes were visualized and imaginedusing Clarity™ Western ECL Substrate (Bio Rad) and LI-CORO system(LI-COR), respectively.

The results of the above can be seen in FIG. 13, with the followingtransfection constructs encapsulated in DMP^(CTx) shown:

Lanes 1, 4 and 7 of FIG. 13: vehicle (mock treated, PBS only),

Lanes 2, 5 and 8 of FIG. 13: mRNA-A (mRNA comprising the sequence forprotein A), and

Lanes 3, 6 and 9 of FIG. 13: mRNA-A-122 constructs (comprising thesequence for protein A and miRNA122, inserted in the variant 1 position,as illustrated in FIG. 3).

Transfection was carried out in healthy hepatocytes (HMCPP5) in lanes1-3, hepatocarcinoma model Hep3B in lanes 7 to 9 and cells from thehepatoblastoma model HepG2 in lanes 4 to 6 as described above, using 0.5μg mRNA-DMP^(CTx) per well. Protein was extracted from each cell line 24hours after transfection. 10 μg of protein was loaded into each lane,and data was taken from two independent experiments. Protein A wasdetected in all tested cell lines when transfected with mRNA-A,indicating that successful transfection was achieved. While transfectedwith mRNA-A-122, translation repression was observed only in healthyhepatocytes, but not in Hep3B and HepG2 cells (lanes 3, 6 and 9),indicating that the miRNA-122 does not fulfil its function in testedliver cancer cells. However, for HepG2 cells transfected withmRNA-A-122, expression of protein A was slightly downregulated comparedto cells transfected with mRNA-A, similar to the pattern previously seenfor mCherry expression, in Example 1. This can be seen clearly in theenhanced exposure photographs (bottom) which show incompletedownregulation in the HepG2 cells. The partial downregulation seen inHepG2 cells further implicates miRNA-122 mediated effects ontranslation, as cells from this line have been shown to retain residualmiRNA-122 activity (Demonstration of the Presence of the “Deleted”MIR122 Gene in HepG2 Cells, PLoS One. 2015; 10(3)).

In summary, modification of 3′UTR mRNA by inserting liver-specificmiRNA-122 target sequence can significantly confine mRNA translation tohepatocarcinoma Hep3B and hepatoblastoma HepG2, but not in normal humanhepatocytes.

Example 3: Oncolytic Viral Combination Therapy in Vitro

It is described herein that the differential expression of provided mRNAconstructs allowed by the method of the invention, and shown in theabove Examples, can be used in combination with oncolytic viral therapy.In particular, where oncolytic viruses have been modified to removevirulence genes, attenuating their replicative ability in healthy cells,the invention can be used to restore the function of those genes, orequivalents thereof, in diseased cells such as cancer cells. Toinvestigate this possibility, the combination of the oncolytic virusHSV-1 (R7041), deficient in US3 (see Leopardi et al, 1997, PNAS 94;7891-7896), and the DMP^(CTx) platform, providing an mRNA constructcoding for US3, and modified with miRNA-122 binding sites, was used in amodel of liver hepatocarcinoma (SEQ ID NO: 4).

General Protocols:

Cell Culture

Human liver hepatocarcinoma (HCC) HepG2 and Hep3B cells were cultured inEagle's Minimum Essential Medium (EMEM, Celigro, USA), 10% FBS,streptomycin (100 μg/mL) and penicillin (100 U/mL-1) as monolayers, at37° C. and in an atmosphere of 5% CO2. HepG2 cells were grown oncollagen coated plates at a collagen concentration of 5 μg/cm2.

Virus Preparation

Frozen R7041 virus was thawed in a water bath at 37° C., and sonicatedfor 30 sec using a bath sonicator (0500 sonicator, Osonica, USA), thentransferred to ice, ready for use.

Toxicity of R7041 Alone Against Human HCC

The US3 mutant R7041 virus is thought to be virtually apathogenic tohealthy cells (Leopardi et al. 1997) and has even shown good safety inimmunodeficient, athymic mice (Liu et al. 2007, Clin Cancer Res 2007;13(19)). To establish a baseline of the efficacy of the R7041 virusagainst liver hepatocarcinoma cells, the model cell lines were treatedwith oncolytic virus alone. Cells from the Hep3B and HepG2 lines wereseeded, in triplicate, into 96-well plates, at 15,000 and 17,000 perwell, respectively.

24 hours later, cells were infected with 3-fold serial dilutions ofvirus, from MOI 0.37 to 0.0001694. 96 hours post-infection, theviability of tested cell lines was measured by MTS assay according tovendor instructions (CellTiter 96® AQueous One Solution CellProliferation Assay, Promega, USA). Absorbance was measured at 490 nmwith a 96-well plate reader (BioTek, Cytation 3, USA). Dose-responsecurves and 50% effective dose values (ED₅₀) were obtained using GraphPadPrism, 7.03.

As shown in FIG. 14, both Hep3B and HepG2 cell lines exhibited similarsusceptibility to R7041, with ED₅₀=0.01 and 0.02 MOI, respectively.However, Hep3B cell lines were seen to be slightly more susceptible toR7041, than were HepG2 cells.

The Combinatorial Effect of R7041 and of mRNA-DMP^(CTx) on Human HCCViability

Prior to evaluation of the combinatorial effect of R7041 virus andmRNA-US3-DMP^(CTx) on human hepatocarcinoma cells, we verified thattransfection of Hep3B and HepG2 cells with mRNA-US3-DMP^(CTx) at 0.04μg/mL mRNA-US3 has no significant effect on cell viability, as measuredby the MTS assay.

Hep3B and HepG2 cells were seeded in triplicate into 96-well plates at15,000 and 17,000 per well, respectively. 24 hours later, cells wereinfected with 3-fold serial dilutions of virus, starting from MOI 0.37up to 0.0001694. Both tested cell lines were transfected twice with afixed dose of 0.04 μg/mL mRNA-US3-DMP^(CTx), 24 and 48 hours afterinfection with R7041, according to the experiment timeline as shown inFIG. 15. Three days post-transfection, viability of tested cell lineswas measured by MTS assay as described above. For both tested humanHCCs, the combination of two different compounds: an oncolytic R7041 anda nontoxic dose of mRNA-US3-DMP^(CTx) (0.04 μg/mL) significantlyenhanced tumor destruction at lower viral titres, as shown in FIG. 16.In this figure, the effect on viability is shown for mRNA-US3-DMP^(CTx)alone at 0.04 μg/mL (y-axis cross), for R7041 alone at various dilutions(grey triangles/diamonds) and for the combination (black circles).

The above Examples indicate that the combination of an attenuatedoncolytic virus with deleted virulence genes, and a supply ofdifferentially expressed replacements for the deleted genes cansignificantly increase the efficacy of oncolytic viral therapy in vitro.In particular, greater effects were seen at lower viral titres when incombination with the composition of the invention.

Example 4: Expression of Delivered Fluorescent Protein mCherry mRNAConstructs in Vivo

In order to determine the applicability of the invention for in vivoapproaches, a mouse model of orthotopic human hepatocellular carcinomawas used. Differential expression of driven by miRNA-122 binding siteshas been shown above (see Example 1) to be applicable in healthy mouseAml12 liver cells in vitro.

Orthotopic Human Hepatocellular Carcinoma (HCC) Model

Animals

Female (CB17/Ics-PrkdcSCID/IcrlcoCrl) Fox Chase SCID mice at 6-8 weeksold, were purchased from Charles River, UK. All in vivo procedures wereapproved by the Subcommittee on Research Animal Care, at CrownBio in UK.

Cells

To generate an orthotopic HCC model, a bioluminescent variant of thehuman Hep3B cell line expressing firefly luciferase (Hep3B-cLuX) wasused. Cells were cultured in EMEM medium (Sigma, UK) supplemented with10% heat inactivated FBS, 2 mM L-Glutamine, 1% NEAA; Cells were treatedweekly with 2 μg/mL Puromycin (Sigma).

Intrahepatic Injection and Tumor Growth Monitoring

Under anesthesia, human Hep3B-cLuX cells (2×10⁶) suspended in 20 μL of1:1 PBS:Matrigel™ were injected in the upper left lobe of the liverusing a 29G needle. The injection site was covered using an absorbablegelatin sponge (AGS), the liver was placed back into the abdominalcavity without disturbing the AGS, and the skin was stitched closed.Tumor growth was checked twice weekly by bioluminescent imaging (BLI).

Briefly, the mice were anesthetised, and 150 mg/kg D-Luciferin wasinjected subcutaneously 15 minutes prior to imaging. BLI image wascaptured and processed using Living Image 4.3.1 software (Caliper LS,US). Mice were weighed three times weekly, or once weekly prior todosing. On the indicated days, the mice were sacrificed, and the liverswere fixed with 2 or 4% paraformaldehyde solution (PFA), before freezingin OCT (Optimal cutting temperature compound—embedding medium) forfurther histopathological analysis.

Formulation of mRNA and Evaluation of Tumor Targeting Efficiency

mRNA sequences comprising the mCherry sequence, and the sequence ofmCherry comprising miRNA-122 (SEQ ID NO: 3) were formulated as describedabove in the ‘Synthesis of DMP^(CTx) and formulation of mRNA’ paragraph,and Table 4. To evaluate selective tumor targeting and the sparing ofnon-diseased liver cells, formulated mRNA was injected into the tailvein of mice bearing orthotopic liver cancer. Briefly, 2×110 of humanHep3B-cLuX cells suspended in 20 μL of 1:1 PBS:Matrigel™ were injectedin the upper left lobe of the liver as described above. Tumor growth wasthen monitored by BLI imaging, also as above. Eight days later, when thetumor was established (BLI≥6×10⁶), 20 μg of formulatedmRNA-mCherry-DMP^(CTx), mRNA-mCherry-122-DMP^(CTx) ormRNA-A-122-DMP^(CTx) per mouse was injected through the tail vein,leading to the delivery particles being taken into the liver byreturning blood flow. Twenty-four hours later last BLI was performed,mice were euthanised, and the livers were excised and imaged by BLI exvivo at the localised liver lesions.

Histology

Briefly, following ex vivo imaging, left liver lobes with tumor wereremoved, fixed with 2% PFA, immersed in 30% sucrose solution (in PBS;pH7.4) at 4° C., embedded in OCT and frozen in isopentane pre-cooledwith dry ice bath, and then stored at −80° C. 5 μm frozen sections(Leica CM300, USA) were subjected to nuclear counterstain with DAPI(VECTASHIELD, Vector Laboratories, USA) or H&E (hematoxylin end eosin)staining. Tumor targeting was assessed by determination of mCherry vsmCherry-122 expression level in tumor and healthy liver usingfluorescence microscopy and/or software. Tumourous and healthy tissuewas determined by H&E staining.

An example of tumor growth monitored by BLI imaging on mock andmRNA-mCherry-DMP^(CTx), and mRNA-mCherry-122-DMP^(CTx) treated mice. Theanimals were injected subcutaneously with D-luciferin, and imaged 15 minlater. Signal was present in the midsection of the animals only as shownin FIG. 17 (A) (upper panel), animals shown are prior to treatment withcompositions. All animals with similar intensities in the midsectionwere dissected, and the livers were imaged ex vivo, FIG. 17 (A) (lowerpanel). The left lobe of liver with tumour was sectioned andcounterstained with DAPI. Fluorescence microscopy was used to determinethe expression of mCherry in healthy liver cells, and in liver tumourcells, 24 hours after injection of formulated mRNA, as shown in FIG. 17(B). mCherry fluorescence was detected in healthy hepatocytes when micewere treated with mRNA-mCherry-DMP^(CTx) (FIG. 17B, middle panel). Whentreated with mRNA-mCherry-122-DMP^(CTx) (Variant 1), translationrepression was observed (FIG. 17B, left panel) FIG. 17B shows healthyliver cells from (left to right), mock treated, mRNA-mCherry-DMP^(CTx),and mRNA-mCherry-122-DMP^(CTx) mice.

In conclusion, the compositions of the invention can be administered invivo and can successfully transfect targeted liver cells. When modifiedwith miRNA binding sites, differential expression can be achieved innon-diseased and tumoural cells.

Example 5: Differential Expression of Delivered US3 mRNA Construct inVivo Between Non-Diseased and Diseased Tissue in the Liver

The in vivo mouse model described in Example 4 was applied toadministration of a delivery particle composition comprising a US3 mRNADMP^(CTx) miRNA-122 construct Differential expression of US3 in thelivers of mice containing Hep3B human cancer was analysed usingimmunohistochemistry with an anti US3 polyclonal antibody. The resultsare shown in FIG. 18, where it can be seen that there is a visibledifference in US3 protein levels between the tumour (darker staining)and non-diseased cells (lighter staining). The differential expressiontracks the boundary of the tumour, as independently verified by apathologist. It can be concluded, therefore, that the compositions ofthe invention can successfully drive differential expression of apotential therapeutic enhancement factor in vivo in a mammalian subject

Immunohistochemistry

Fresh frozen sections were cut at 5 μm (microns) and air-dried forapproximately one hour prior to fixation with 4% paraformaldehyde atroom temperature (RT) for 15 minutes. Sections were washed in runningtap water and transferred to PBS-0.1% Tween. Sections were incubatedwith 2.5% normal horse serum (ready to use, ImmPRESS HRP anti-rabbit IgGperoxidase polymer detection kit, Vector MP-7401) for 20 minutes. Theslides were drained and incubated with primary antibody to US3 diluted1:400 (Acris AP55266SU-N). The antibodies were diluted with PBS-0.1%Tween and negative controls were included where the primary antibody wasomitted and slides incubated with the antibody diluent PBS-0.1% Tweenfor one hour at RT. Slides were washed with PBS-0.1% Tween andendogenous peroxidase blocked with 0.3% hydrogen peroxide diluted withelga water for 10 minutes. Slides were washed with PBS-0.1% Tween andincubated with ImmPress anti-rabbit IgG reagent (ready to use, ImmPRESSHRP anti-rabbit IgG peroxidase polymer detection kit, Vector MP-7401)for 30 minutes at RT. Slides were washed with PBS-0.1% Tween andincubated for 5 minutes with chromogen ImmPACT DAB (ImmPACT DABPeroxidase (HRP) Substrate, Vector SK-4105) then washed with elga waterand counterstained as appropriate with Mayer's haematoxylin. A furtherwash in elga water briefly was carried out and blue in running tap waterfor 5 minutes. The slides were dehydated, cleared and mounted (95% IMS,99% IMS x2 and xylene x2) then covered with a coverslip.

Although particular embodiments of the invention have been disclosedherein in detail, this has been done by way of example and for thepurposes of illustration only. The aforementioned embodiments are notintended to be limiting with respect to the scope of the appendedclaims, which follow. It is contemplated by the inventors that varioussubstitutions, alterations, and modifications may be made to theinvention without departing from the spirit and scope of the inventionas defined by the claims.

The invention claimed is:
 1. An isolated mRNA sequence comprising: atleast one coding sequence which codes for the at least one polypeptide,wherein said polypeptide is an oncolytic virus gene product involved inviral genome replication, or a mammalian cellular equivalent orhomologue thereof; at least a first untranslated region (UTR) sequence;at least three substantially different micro-RNA (miRNA) binding sitesequences, wherein at least two of the at least three miRNA binding sitesequences are configured to bind miRNAs differentially expressed withindifferent cell types comprised within a target organ of a subject,wherein the organ is liver; and wherein the at least three substantiallydifferent miRNA binding site sequences are located within the first UTRsequence.
 2. The isolated mRNA sequence of claim 1, wherein the mRNAsequence comprises greater than three miRNA binding site sequences. 3.The isolated mRNA sequence of claim 1, wherein the different cell typescomprised within the liver of the subject are selected from the groupconsisting of: non-neoplastic cells; a transformed cell phenotype; apre-cancerous phenotype; and a neoplastic phenotype.
 4. The isolatedmRNA sequence of claim 3, wherein the liver cell type is a pre-cancerousor neoplastic cell type.
 5. The isolated mRNA sequence of claim 4,wherein the liver cell type is a liver cancer selected from a primarycancer or secondary/metastatic cancer in the liver.
 6. The isolated mRNAsequence of claim 5, wherein the liver cancer is a cholangiocarcinoma;angiosarcoma, hepatocarcinoma or hepatoblastoma.
 7. The isolated mRNAsequence of claim 1, wherein the mRNA comprises a further codingsequence that encodes a therapeutic enhancement factor.
 8. The isolatedmRNA sequence of claim 7, wherein the therapeutic enhancement factor isselected from the group consisting of a tumor suppressor protein, aprogrammed cell death protein, an inhibitor of a programmed cell deathpathway, a monoclonal antibody or fragment or derivative thereof, asequence-specific nuclease, an oncolytic virus gene product, a cytokine,a chemokine, a fluorescent marker protein, an immunomodulatory moleculeand combinations thereof.
 9. The isolated mRNA sequence of claim 1,wherein the at least three substantially different miRNA binding sitesequences hybridize with an miRNA selected from the group consisting ofmiRNA-122; miRNA-124a; miRNA-125; Let-7; and miRNA-375.
 10. The isolatedmRNA sequence of claim 1, wherein the oncolytic virus gene product is aviral gene product of an oncolytic virus selected from of the Groups Iviruses of the Baltimore classification of viruses or mammalianequivalent or homologue thereof.
 11. The isolated mRNA sequence of claim1, wherein the oncolytic virus gene product is a ribonucleotidereductase.
 12. The isolated mRNA sequence of claim 1, wherein the mRNAcomprises a further coding sequence.
 13. A pharmaceutical compositioncomprising: at least one mRNA sequence comprising: at least one codingsequence which codes for at least one polypeptide, wherein saidpolypeptide is an oncolytic virus gene product involved in viral genomereplication, or a mammalian cellular equivalent or homologue thereof; atleast a first untranslated region (UTR) sequence; at least threesubstantially different micro-RNA (miRNA) binding site sequences,wherein at least two of the at least three miRNA binding site sequencesare configured to bind miRNAs differentially expressed within differentcell types comprised within a target organ of a subject, wherein theorgan is liver; wherein the at least three substantially different miRNAbinding site sequences are located within the first UTR sequence; and apharmaceutically acceptable carrier.
 14. The isolated mRNA sequence ofclaim 1, or the pharmaceutical composition of claim 13, wherein theoncolytic virus is selected from the group consisting of VesicularStomatitis Virus, Maraba virus, Polio virus, Reovirus, Measles virus,Newcastle disease virus, Coxsackievirus A21, Parvovirus, Herpes SimplexVirus Type 1, and Adenovirus.
 15. The pharmaceutical composition ofclaim 13, wherein the oncolytic virus gene product is a ribonucleotidereductase.
 16. The pharmaceutical composition of claim 13, wherein theisolated mRNA sequence is encapsulated in a delivery particle.
 17. Thepharmaceutical composition of claim 13, wherein the delivery particlescomprises aminoalcohol lipidoids.
 18. The pharmaceutical composition ofclaim 13, further comprise one or more targeting agents selected fromthe group consisting of proteins, peptides, carbohydrates,glycoproteins, lipids, small molecules and nucleic acids.
 19. Thepharmaceutical composition of claim 13, wherein the liver cell type is apre-cancerous or neoplastic cell type.
 20. The pharmaceuticalcomposition of claim 13, wherein the liver cell type is a liver cancerselected from a primary cancer or secondary/metastatic cancer in theliver.
 21. The pharmaceutical composition of claim 13, wherein the livercancer is a cholangiocarcinoma; angiosarcoma, hepatocarcinoma orhepatoblastoma.
 22. The pharmaceutical composition of claim 13, whereinthe composition is formulated for intrahepatic delivery.